- Email: [email protected]
Alpha-fetoprotein signal sequences: a proposed mechanism for subcellular localization and organelle targeting
J. theor. Biol. (1995) 176, 103–113
Alpha-fetoprotein Signal Sequences: A Proposed Mechanism for Subcellular Localization and Organelle Targeting G. J. M Laboratory of Human Genetics, Wadsworth Center for Lab & Research, NYS Dept. of Health, Albany, New York 12201, U.S.A. (Received on 26 October 1994, Accepted in revised form on 17 April 1995)
Alpha-fetoprotein (AFP) is an oncofetal protein, classified in an ‘‘albuminoid’’ superfamily (with albumin and Vitamin-D binding (Gc) protein) comprising molecules with three characteristic globular domains. The cellular uptake and internalization of AFP and its subcellular compartmentalization has previously been reported in a multitude of cell types. Studies have also emerged which have detected and characterized binding proteins complexed to AFP in various cell membranes and intracellular compartments. However, the literature is devoid of any attempts to relate these binding proteins to possible intracellular trafficking interactions of AFP. Recombinant DNA mutation/deletion technology has provided a means to pinpoint the amino acid sequence location of organelle localization signals on various transcription factors and/or receptors. Several subdomain regions on AFP have been reported to mimic heptad dimerization regions of the steroid/thyroid nuclear receptors. In light of these transcription factor-like docking motifs reported for AFP, the present report purposes various subdomain regions which might constitute basic amino acid sequences resembling recognition signals for binding (dimerizing) proteins. AFP appears to possess multiple prototypic amino acid sequence cassettes on each domain which consist of (i) classical short, compact sequences found on both steroid and thyroid receptors; (ii) proto-signals resembling a steroid receptor type; and (iii) degenerative signal sequence similar to the thyroid/retinoic acid receptor type. The concepts identifying binding or escort proteins for AFP together with organelle signal sequences on AFP have been reconciled in a composite hypothesis to formulate a mechanism which could account for some of the growth-regulating properties described for AFP during the last decade. 7 1995 Academic Press Limited
During the last 20 years, a plethora of studies has emerged implicating the existence of AFP in a form complexed with other proteins or peptides (see review, Mizejewski, 1994). This bound form of AFP is found in both sera and tissue extracts and is dissociable by the use of high salt (0.4 M KCl) or serine protease inhibitors (Sarcione et al., 1983; Mizejewski & Brown-Biddle, 1989). To date, the physiological significance of this bound-form of AFP has eluded clarification. Earlier studies of AFP function documented that this fetal protein displayed serum binding/transport properties regarding fatty acids, steroids, retinoic acid, heavy metals, and various drugs (Tomasi, 1977). More recent studies have demonstrated that AFP is capable of both oncogenic and ontogenetic growth regulation,
Introduction During ontogenic development in mammals, products termed oncofetal or phase-specific proteins are produced at various stages of embryonic, fetal and neoplastic growth (Tatrinov, 1965; Abelev, 1971). One of the most widely studied onofetal proteins to date is alpha-fetoprotein (AFP), an alph-1glycoprotein comprised of a single-chain 70 kDa polypeptide containing 3–5% carbohydrate (Mizejewski, 1985; Deutsch, 1991). AFP is also produced and secreted in patients with hepatocellular carcinomas, teratoblastomas, hepatitis, and cirrhosis (Crandall, 1981). Thus, the presence of circulating AFP in serum levels renders it a useful marker for fetal distress, tumor growth and hepatic dysfunction. 0022–5193/95/170103+11 $12.00/0
103
7 1995 Academic Press Limited
104
. .
F. 1.—caption opposite.
/ and that such modulation might be attributed to transcriptional enhancement and/or suppression activities (Mizejewski & Warner, 1989; Mizejewski et al., 1990). It has also been documented that AFP displays both cellular uptake and transmembrane passage via specific cell surface receptors (Laderoute, 1991; Moro et al., 1993) and cytoplasmic binding proteins culminating in either receptosome or lysosomal pathways (see review, Mizejewski, 1994; Suzuki et al., 1992). It may be hypothesized that AFP, once endocytosed, might be capable of diverse subcellular localization by means of amino acid signal-like sequences which could affect organelle targeting. In the present treatise, the reports involving the uptake of AFP, the bound forms of AFP, findings of presumptive amino acid signal sequences, and the observations of growth regulatory properties of this oncofetal protein have been merged to propose a mechanism implicating these AFP activities with intra-cellular localization and organelle targeting. Intracellular Organelle Targeting During the period in which AFP-binding proteins were observed and reported, the functional domains of the steroid/thyroid nuclear receptor superfamily members were being characterized using recombinant DNA deletion/mutation technology (Evans, 1988; Ham & Parker, 1989). This superfamily of nuclear receptors are known to be inducible transcriptional regulators which bind steroid/thyroid/vitamin ligands and recognize hormone response elements of target genes to enhance or repress transcription (Wahli & Martinez, 1991). The functional domains of these receptors has been assigned the letters A to F in which C is the DNA binding domain and E is the ligand binding domain (Forman & Samuels, 1990). The D domain has not been as extensively characterized but is thought to contain a major nuclear localization signal (NLS) which serves as a targeting site to transport the receptor into the nucleus (Gronemeyer, 1992). However, other NLS sites have also been identified in the C and E domains and appeared to
105
function in concert or in a cooperative fashion to enact organelle translocation (Ylikomi et al., 1992; Dang et al., 1989). Although a consensus sequence has not yet been ascertained, the targeting signals are thought to consist of three types: (1) a concise, compact stretch of basic amino acids (usually 3–7 residues); (2) two or more weaker and less concise proto-signals containing clusters of basic amino acids interspersed with neutral amino acids; and (3) multiple degenerative sequences which are extended in length, noncontiguous, scattered residues of basic amino acids (Roberts, 1989; Garcia-Bustos et al., 1989). The compact amino acid sequence signals are frequently flanked by either glycine, proline or acidic amino acids (Silver, 1991). It has been proposed that the presence of neutral amino acids flanking the NLS provide insulating boundaries for the signal site or serve other modulating functions (Stochaj & Silver, 1992). Proteins bearing such signal amino acid sequences are known to bind a cytoplasmic signal recognition receptor (55–75 kDa binding proteins) which then escorts the signal bearing protein to various target organelles (i.e. nuclear pore complex, outer mitochondrial pore, etc.) (Adam et al., 1989; Schatz, 1993). Figure 1 represents a compilation of the various NLS’s found in various members of the steroid/ thyroid/vitamin receptor superfamily (Picard et al., 1990; Dang et al., 1989; Guiochon-Mantel et al., 1989); these were then compared to presumptive signal amino acid cassette sites proposed for the AFP molecule. It became immediately apparent that the AFP molecule itself displayed intrinsic prototypic signal-like sequence homologies among its own three domains. There further appeared to be multiples of the different signal types on all three domains of AFP. First, four compact type of signal-like cassettes present on all three domains of AFP appeared to resemble those of the thyroid/retinoic acid receptors more than the steroid receptor family members, although both share similarities.
F. 1. Amino acid sequence alignment of the various organelle localization signal cassette sites proposed for rodent and human alpha-fetoprotein versus the established sites on the steroid/thyroid related receptors. The amino acid (AA) sequences of the various classes of organelle targeting signals are listed for both mouse (M) and human (H) alpha-fetoprotein and the nuclear receptor superfamily members: (a) Class I localization signals are compact, concise localization sites consisting of three to seven basic residues shown with surrounding AAs. (b) Class-II type localization proto-signal sites comprised two or more weaker and less concise acidic AA stretches in a bipartite fashion. (c) Class-III type localization degenerative sequences are extended in length, non-contiguous, with scattered residues of basic AAs. AFP, alpha-fetoprotein, AR, androgen receptor; ER estrogen receptor; GR, glucocorticoid receptor; MR, mineralcorticoid receptor; PR, progesterone receptor; RAR, retinoic acid receptor; RXR, retinoic acid-X-receptor; VDR, Vitamin D receptor. All receptors denote the human species; T3R represents the B-isoform, while RAR and RXR, the a-isoforms.
106
. .
Second, two signal sites within the steroid binding domain bore resemblance to comparable bipartite proto-signal sites reported for the steroid nuclear receptor members (LaCasse et al., 1993). Third, two of the localization sites appeared to comprise part of a more degenerative signal sequence (Horowitz et al., 1989), one site of which lies on the second domain of AFP. Among steroid receptor superfamily members, cytoplasmic receptor residence has been noted for the glucocorticoid, mineralcorticoid, androgen and the vitamin-D receptors (Simental et al., 1991; Carlberg et al., 1993). For purposes of comparison, the glucocorticoid receptor provides a good cytoplasmic example since the estrogen and progesterone receptors are thought to reside principally in the nucleus, while the glucocorticoid receptor has been localized in nonnuclear cytoplasmic fractions (Guiochon-Mantel et al., 1989). The glucocorticoid receptor contained a major NLS positioned at the carboxy terminal end of the second zinc finger in the D-domain (termed NL1); an NL2 region is located in the hormone (ligand) binding region of the E-domain (Ylikomi et al., 1992; Picard et al., 1990). The NL1 would correspond to the D-domain of most steroid receptors and might be exemplified on AFP as amino acid sequences 339–354 in which a series of basic compact, concise amino acids reside at 341–345. The NL2 region of the glucocorticoid receptor contains two subregions that require hormone-binding for activation, one located toward the amino-end of the ligand binding domain and the other at the carboxy-terminal last third of the domain. These two NLS regions on the glucocorticoid receptor might be analogous to the proposed protosignal amino acid sequences 416–431 and 465–478 on AFP. In the glucocorticoid receptor, these regions of the ligand binding domain required hormone for activation; thus, it may be more than coincidence that AFP amino acid sequences 416–431 and 465–478 lie at the beginning and end of the estrogen binding subdomain reported for rodent AFP (Nishi et al., 1991). The latter proto-signal which appeared to be present at AFP amino acids 465–478 lies directly in a region crucial to steroid (estrogen) binding regulation in the third domain of AFP (Nishi et al., 1991). These two regions on AFP could serve as potential proto-signals for organelle (nuclear) accumulation as reported for the glucocorticoid, estrogen and progesterone receptors (LaCasse et al., 1993; Picard et al., 1987). A concise signal in the third domain of human AFP might be located within amino acids 537–562 (532–545 in Fig. 1) which lie in a strongly hydrophobic region of leucine zipper-like heptad repeats resembling
a stretch of 100 amino acids comprising a large putative dimerization subdomain found in the nuclear receptor superfamily (Mizejewski, 1993). It has been demonstrated that a strongly hydrophobic carboxy terminus sequence can override a NLS independently of the amino acid context of the signal-bearing protein in the cytoplasm (Zee et al., 1991). Upon binding E2, the rat uterine estrogen receptor has been reported to undergo a conformational change exemplified by a dramatic decrease in surface hydrophobicity localized toward the carboxy-terminal side of the steroid binding domain (Fritsch et al., 1992). In comparison, AFP is known also to undergo a conformational change in the presence of high E2 concentrations, as evidenced by a change in the UV difference spectrum (Jacobson et al., 1989). This time-dependent absorbance maximum of AFP displayed a high molar extinction coefficient suggesting a positional change in intrinsic aromatic amino acids. A conformational change on AFP, as a result of E2 exposure results in a dramatic change in carboxy terminus hydrophobicity, which could have relieved a hydrophobic override in the signal site and exposed the amino acid localization sequence for subsequent organelle targeting. Similar intramolecular masking effects has been reported for several other proteins including NF-kb, c-Rel, dorsal and the glucocorticoid receptor (Whiteside & Goodbowen, 1993), and could apply to AFP (Table 1) in light of recent studies demonstrating AFP regulation of estrogen-induced uterine and tumor growth (Mizejewski et al., 1983; Jacobson et al., 1990; Mizejewski et al., 1990). Finally, molecular masking is also thought to occur via small ligand molecules (peptides, organic molecules, protease inhibitors/substrates, etc.) binding at or overlapping onto organelle cassette signal sites (Roberts, 1989; Silver, 1991). Studies with synthetic peptide fragments bound to the LBD as co-transporters have confirmed such findings in the steroid receptor superfamily (Dalmon et al., 1991). Unlike the glucocorticoid receptor, estrogen and progesterone receptors are known to reside largely as nucleoplasmic receptors and display a different array of NLS’s (Picard et al., 1990). The estrogen and progesterone receptors contain multiple proto-signals (pNLS) utilized for nuclear targeting (none of which suffices on its own) which cooperate or act in concert as a result of a ligand-induced conformational assemblage. The estrogen receptor contains an estrogen-induced pNLS in addition to three other basic amino acid-rich proto-signals which cooperate following exposure to estrogen (Ylikomi et al., 1992). Thus, AFP might mimic the estrogen receptor in displaying multiple weaker signal-like sites (amino
/
107
T 1 Various cellular transcription factor and/or DNA binding proteins and their respective inhibitors DNA binding protein/complex 1. Steroid/thyroid receptors (complex) 2. NF-kB complex (rel-Family) 3. AP-1, activator protein-1 4. MyoD
Related or component subunits
Type of oligomer formation
Apparent subcellular residence
ER, PR GR, MR AR, RAR RXR, TR, RAR p50 p65 p50 p52 C-JUN C-FOS C-myc E12 E47
Monomers Monomers HomoHeterodimers Heterodimers
Cytoplasm and nuclear
HSP-90 HSP-70 HSP-59
Activation, inhibition
Cytoplasm
Heterodimers
Nucleus (cytoplasm) Cytoplasm
lkB (43 kDa) BcL-3 (46–56 kDa) 1P-1 (37 kDa) 1D (43 kDa)
Activation, inhibition Activation (enhancement) Enhancement, inhibition Enhancement, inhibition
HomoHeterodimers HomoHeterodimers
Cytoplasm
Family of inhibitors
Transcriptional regulation
Reference Smith, 1993 Cadepond, 1991 Rexin, 1991 Dalmon, 1991 Shyamala, 1992 Gilmore, 1993 Wulczyn, 1992 Lopez, 1993 Avwerx, 1991 Lasser, 1989 Davis, 1990
Such factors could serve as prime candidates as the cytoplasmic alpha-fetoprotein binding proteins. These all represent cellular immediate-early response gene products, many of which utilize protein kinase C (PKC) signal transduction pathways following cell stimulation by serum factors and/or phorbol esters (tPA, 12-0-tetradeconyl-phorbol-13-acetate). The present report proposes that AFP binding to the transcription factor (DNA-binding protein) would result in growth inhibition, while AFP complexing to the inhibitor would cause growth enhancement. Abbreviations used: ER, estrogen receptor; erbA, putative thyroid hormone receptor proto-oncogene products; MR, mineralcorticoid response element on DNA; RAR, retinoic acid receptor; RXR, retinoic-X-receptor, T3R, thyroid hormone (triiodothyronine) receptor; VDR, vitamin D receptor.
acids 416–431, 465–478). A fourth suspected NLS of the estrogen receptor, although more degenerative, could exist toward the amino terminal side of the DNA binding domain. Such discontinuous motifs are known to exist in other superfamily members (such as retinoic acid, thyroid, retinoic-X and mineralocorticoid receptors, etc.) (La Casse et al., 1993; Horowitz et al., 1989), and AFP may indeed have such a weak NLS-like site at amino acid sequences 223–237. Other degenerative (weak) presumptive sites on the first domain of AFP at amino acids 80–94, 102–116 and 142–156 could also serve as presumptive signals for other organelle targeting. Frequently, the signals toward the amino-terminal portion of other proteins (i.e. prepeptide signal piece) are associated with targeting to organelles such as the mitochondria, Golgi, ribosomes, and endoplasmic reticulum (Roise & Schatz, 1988; Rapaport, 1991), although a lamin receptor contains an amino terminal NLS site (Soullam & Worman, 1993). Thus, these proposed amino-terminal localization-like cassettes on AFP might represent mitochondrial, microsomal or EPRrelated signals (see Class 1, Fig. 1) in contrast to NLS’s. It may be no coincidence that one of the proposed signal cassette sites on AFP (amino acids 532–545) is associated with a presumptive hetero-dimerization region which has previously been purposed for this fetal protein. Since the nuclear targeting escort proteins usually recognize a targeting signal, it would seem reasonable to find (as in AFP) the dimerization sites located in the near vicinity, either
flanking the signal cassette or inclusive with it. This observation would suggest a direct association between the presence of the signal cassette and the location of the dimerization interface. It would be conceivable that NLS sites on proteins also function as dimerization recognition and/or docking sites (Smith & Toft, 1993). Pratt et al. (1993) have proposed that the arginine/lysine-rich sequences comprising the signal site would bind to the escort protein displaying signal docking glutamic/aspartic acid-rich sequences. In this regard, it has already been demonstrated that the signal sequence site on precursor proteins bind to the organelle import receptors intended for mitochondrial targeting (Murakami et al., 1993).
Binding Proteins with NLS Recognition It would seem that AFP may somehow be capable of interacting with nucleocytoplasmic trafficking or organelle targeting since it appeared to contain multiple presumptive signal sites (Fig. 1). Cytoplasmic proteins that specifically recognize and bind organelle signal-proteins have been reported to serve as escort receptors for nuclear import (Adam & Gerace, 1991; Haino et al., 1993). Interaction of NLS-containing receptors with the signal-bearing protein was the initial step in mediated nuclear import. The cytoplasmic binding proteins are reported to range in size from
108
. .
56–76 kDa or greater while the nuclear poreassociated proteins appear larger, approximating 140–185 kDa (Rosen et al., 1991; Sukeqawa & Blabel, 1993). The initial binding of the signal protein to the nuclear membrane is associated with high affinity binding is saturable, dependent upon the presence of cytosol, and is sensitive to inactivation by N-ethylmaleimide (Newmeyer & Forbes, 1990). A subsequent step was an NLS-dependent binding to the cytoplasmic side of the nuclear pore complex (NSC) based on in vivo and in vitro studies using colloidal gold probes (Adam et al., 1989; Adam & Gerace, 1991). Thus, the NPC appeared to serve as a morphological connection between the nuclear and cytoplasmic compartments and could potentially exert a powerful influence on controlling gene activity via transport of regulatory components for the selective interaction with certain transcribed genes (i.e. gene gating; see below). A family of cytoplasmic glycoproteins termed nucleoporins bearing O-linked N-acetylglucosamines have been identified in eukaryotes of which rat nucleoporin p62 is a member (Starr et al., 1990; Loeb et al., 1993). This karyophilic protein consists of three domains of which the carboxy-terminal third domain displays heptad repeats comprising a dimerization domain. It is noteworthy that Laderoute (1991) and Moro et al. (1993), using monoclonal antibodies, identified AFP-binding protein doublet moieties at 62 and 67 kDa in which the binding proteins contained O-linked glycosylation sites as found in other systems (Cordes et al., 1993). Similarly, identification of binding proteins for the NLS site of the glucocorticoid and thyroid-hormone (triiodothyronine) receptors have been reported in lymphoma cytosols as 60 and 76 kDa kinase-related polypeptides (LaCasse et al., 1993). It is tempting to speculate that the binding of at least some of the cytosolic and saturable membrane-associated protein binding to AFP could actually be a class of NLS recognition escort proteins. Alternatively, AFP itself might serve as a NLS recognition binding protein (Table 1). It is of particular interest that non-liganded progesterone receptor monomer was shown to dimerize with the liganded monomer, forming oligomers (dimers) that are transported into and out of the nucleus (Guiochon-Mantel et al., 1989). Thus, the NLS receptors themselves might function to shuttle receptors and nucleoproteins between the cytoplasm and the nucleus in a bidirectional fashion. One could speculate that liganded AFP could have interacted with non-liganded AFP to help explain the results of some previous experiments where E2-treated AFP was capable of growth regulation in normal and
neoplastic reproductive tissues (Jacobson et al., 1990; Mizejewski et al., 1990). It is further conceivable that AFP, when exposed to a high concentration of ligands, converts AFP into a signal sequence binding protein, which could recruit non-liganded proteins (i.e. AFP?) transcription factors, or steroid receptors (Table 1) as its dimer partner for cytoplasmic translocation toward the organelle surface (nuclear) (Panels II & III, Fig. 2) or inhibition of that transport. Transorganelle (transnuclear) passage involves interaction of the signal site bearing protein with a putative transporter protein assembly situated in or about the central channel of the NPC (Loeb et al., 1993; Cordes et al., 1993). Translocation of the import protein through the central channel of the NPC requires ATP phosphorylation and is accompanied by size-selective gating at the channel pore (Wente et al., 1992; Radu et al., 1993). This phosphorylation step is inhibited by reduced temperatures and by wheat germ agglutinin lectin. Pore complex proteins of 153 kDa (180 kDa in SDS–PAGE), which have been cloned and sequenced, contained zinc fingers, bound DNA, and faced the nucleoplasmic interface (Sukegawa & Blobel, 1993). It is thought that the 153 kDa could be a DNA binding subunit of the NPC that could link the complex to cognate DNA sequences thereby gating transducable genes to the pore complex. It is interesting that Yamasaki, et al. (1989) also identified a 140 kD NLS binding protein that associated with rat liver nuclei. As previously reported, an AFP-binding protein of 185 kDa (SDS) has been detected in the solubilized membrane fraction from breast cancer cells (Laderoute, 1991). Reaction of monoclonal antibodies to the AFP-binding protein further resulted in phosphorylation of this protein involving ATP release. This protein could be likened to the 153 (185) kDa protein requiring ATP phosphorylation as described above. Is AFP a Heat Shock Protein? One of the most interesting properties of HSP-70 is its ability to form complexes with a variety of viral oncogene products (Yehiely & Oren, 1992). In fact, it has been proposed that heat shock proteins function as cross-linking agents, especially cytoskeletal (actin) elements, to facilitate physiological cascade phenomenon (Tsang, 1993). In a similar fashion, the c-erbA dimerization sites proposed for AFP in a previous study has been advanced in attempting to explain the regulation of growth by AFP as reported in previous investigations (Mizewjewski, 1993). It is conceivable that AFP could function as a heat shock cognate-like
/
109
F. 2. The uptake, transmembrane passage, and metabolic fate of liganded and free AFP are depicted in an epithelial cell model depicting four possible metabolic pathways for AFP. The cell is subdivided into four panels (I–IV). Panel I depicts both liganded and non-liganded AFP and Albumin (ALB) endocytosis which has been documented in the literature. Panel II proposes that AFP be liganded displaying partial signal site exposure and conformational alteration leading to an endocytotic pathway involving endoplasmic reticulum (EPR) entry toward the nuclear pore. Panel III displays a similar vesicular uptake of AFP, except that AFP is released from the endosome directly into the cytoplasm. Panel IV portrays an endocytotic liganded pathway in which AFP is also released directly into the cytoplasm or could apply to AFP synthesized but not secreted from the cell. CMR, cell membrane receptor; DBP, DNA-binding protein; EL, endolyzozome, EPR, endoplasmic reticulum; ER, estrogen receptor; EV, endosomal vesical; GC, Golgi complex; HRE, hormone response element; HSP, heat shock protein; N, nucleus; NBP, nuclear binding protein; RP, receptor-binding protein.
protein or an accessory protein and bind to the nuclear receptor complex at their heat shock protein designated sites or perhaps dimerize or compete with the heat shock protein itself. It has been reported that the HSP-90 protein appears to cap the steroid receptor DNA-binding region, preventing it from binding to the DNA hormone response element thereby stabilizing the receptor in the cytoplasm (Cadepond et al., 1991). Dimerization of heat shock protein to AFP could render the steroid nuclear receptor more susceptible to degradation and possibly disrupt kinase activities. Alternatively, the heat shock protein and/or AFP could either mask a signal region or interfere with the localization recognition site which is linked to receptor activation since
phosphorylation was found coincident with HSP-90 dissociation from the 9S glucocorticoid receptor complex following a conformational change (Orti et al., 1993; Csermely et al., 1993). It has been reported that albumin synthesis could be induced by heat shock stimulation in fetal rats (Srinivas et al., 1987); however, heat shock stimulation of AFP resulted only in an unstable, degraded AFP mRNA while circulating AFP was unaffected (Srinivas & Revathi, 1994). Since albumin has recently been shown to bind the ATP molecule (Bauer et al., 1992), the binding of ATP to AFP is also possible based on crystal structure molecular similarities. Although, AFP has not been reported to exhibit amino acid sequence homology with the heat shock family of proteins, it is
. .
110
evident that AFP competition, interaction or dimerization with the heat shock proteins have yet to be ruled out. To date, present data would argue against AFP as a heat shock protein if elevated temperature (heat) is used as the criteria. However, glucose-shocked AFP has yet to be studied since glucose levels are known to correlate with circulating AFP levels in the fetus; pregnant women with insulin-dependent diabetes are also known to have lower AFP serum levels than non-affected pregnant women (Martin et al., 1990; Rule et al., 1981). If it were possible to link a heat shock protein-like polypeptide binding function to AFP, then a myriad of additional molecular chaperone roles could be implied. One or more of these functional roles might involve polypeptide chain folding, phosphorylation, maintenance of inactive state for transcription factors, nuclear translocation signalling, cross-talk cell signalling, induction of conformational change (as in receptor complex dissociation), transmembrane passage, regulation of proteolytic degradation of activated receptors, and binding to the heavy chain of IgG (Rothman, 1989). It is noteworthy that AFP has been detected in a form complexed to both immunoglobulin G and M and to serine protease substrates/inhibitors (Mizejewski & Brown-Biddle, 1987). Thus, it is conceivable that AFP could be a heat shock protein-like prototype of the adult stress/growth proteins expressed during the embryonic and fetal life of the organism in which dimerizing to polypeptides may be one of the colligative properties of this protein. AFP Metabolic Pathway Models The above hypothesis stated that AFP might display potential multiple AA sequences which resemble organelle targeting signal sites reminiscent of steroid/ thyroid nuclear receptors. Such sequences could also provide recognition sites to attract cytoplasmic receptor and/or transcription factors for binding or dimerization. Following transmembrane passage of AFP, which has been well documented (Uriel, 1984; Naval et al., 1985), four cell model pathways may be postulated for the metabolic fate of this fetal protein depending on its conformational state (Fig. 2). The first pathway (panel I, Fig. 2) involves a welldocumented shuttle system for endocytosed AFP free or liganded to fatty acids and/or steroids which permits ligand release and recyclization of AFP to the extracellular compartment via the Golgi complex (Uriel et al., 1986; Mizejewski et al., 1989). In a second pathway (panel II, Fig. 2), the cellular uptake of ‘‘partial’’ conformationally altered AFP (incomplete
signal unveiling following exposure to excessive ligand concentrations) might involve receptosome transport to the perinuclear space of the endoplasmic reticulum for possible protein-to-protein interaction. AFP has been histochemically localized within the perinuclear spaces (Hajeri-Gemond et al., 1985); such regions are known to be continuous with channels of the endoplasmic reticulum. A third pathway, involving ligand-induced ‘‘complete’’ conformationally-altered AFP, could involve endosomal release of AFP directly into the cytoplasm (Panel III, Fig. 2). The endosomal release could be related to the dissolving of clathrin coatings reminescent of heat shock protein functions. Conformational transformation could expose full signal recognition sequences on AFP and induce a subsequent binding to cytoplasmic signal recognition escort proteins for import to the nucleus via the nuclear pore in direct contact with cytoplasm. Once within the nucleus, AFP might either dimerize with various members of the steroid/ thyroid receptor superfamily to form inactive heterodimers, bind to repressor/ inhibitory factor targeted for transcription enhancement, or bind nuclear cofactors (i.e. API, RXR, NF-kb, FOS/JUN; Table 1) in transit for transcriptional regulation (Lopez et al., 1993; Whiteside et al., 1993). As a fourth cell model pathway, one cannot rule out the possibility that AFP could bind and retain karyophilic transcription factors and/or their inhibitors in the cytoplasm, thus preventing their import into the nucleus (Panel IV, Fig. 2). This could also apply to AFP synthesized in a cell but not secreted (Sarcione et al., 1976). Thus, by modifying the activities of receptor-associated factors, AFP might function to inhibit translocation before signal (transcription) activation could occur (Panel IV, Table 1). NF-kb nuclear transcription factor, which is found in both lymphoid and uterine cells, is related to the c-rel oncogene and provides a prime example of such DNA-binding factors (Wulcyzn et al., 1992). NF-kb, a 50/65 kDa hetero-dimer, is normally retained in the cytoplasm by a 36 kDa inhibitor termed I-kb and is released upon phosphorylation and/or estrogen stimulation (Shyamela & Guit, 1992). In this regard, it is of great interest that AFP binding proteins of 62–67 kDa mass have been reported (Laderoute, 1991; Moro et al., 1993). Another example, the glucocorticoid receptor, is retained in the cytoplasm complexed to HSP-90 (Rexin et al., 1991). Competing off the heat shock protein from the receptor complex could also result in glucocorticoid receptor confinement to the cytoplasm. In both instances, the protein bound in a complex is unable to bind DNA since their NLS’s are concealed in both the former and latter examples, preventing
/ nuclear import. It is also possible that AFP could bind the cytoplasmic inhibitors of the DNA binding proteins (i.e. IkB or IP-1), allowing subsequent translocation of the nuclear factor to occur (see Fig. 2). Such a mechanism could provide the rationale to account for the growth enhancement previously attributed to AFP. In contrast to the AFP growth inhibition described in the above paragraph, this growth enhancement could occur with AFP whose signal sequences are concealed due to insufficient (incomplete) conformational exposure of the various signal cassettes, the proto-NLS, or the degenerative signal sites (Panel IV, Fig. 2). In Panels III and IV of Fig. 2, AFP need only to reside in the cytoplasm to produce its effect on growth. Concluding Remarks Based on this NLS-binding protein concept together with binding proteins described in previous reports, AFP could serve to either inhibit or enhance gene regulation at the transcriptional level, as previous experimental findings would seem to indicate (Mizejewski et al., 1990; Jacobson et al., 1990). The volume of evidence in the literature supporting AFP as a factor in growth regulation can no longer be ignored. It is hoped that the present report will both arouse interest and provide a working hypothesis to help explain the growth-related properties of AFP to the biomedical community.
REFERENCES A, G. I. (1971). Alpha-fetoprotein in association with malignant tumors. Adv. Cancer Res. 14, 295–357. A, S. A. & G, L. (1991). Cytosolic proteins that specifically bind nuclear location signals are receptors for nuclear import. Cell 66, 837–847. A, S. A., L, T. J., M, M. A. & G, L. (1989). Identification of specific proteins for nuclear location sequence. Nature, Lond. 337, 276–279. B, M., B, J. & T, W. E. (1992). ATP binding to bovine serum albumin. FEBS Lett. 313, 288–290. C, F., S-G, S-M, I., J, H, S. M., G V., E, R. M. & B, E. E. (1991). Heat shock protein-90 as a critical factor in maintaining glucocorticoid receptor in a non-functional state. J. biol. Chem. 266, 5834–5841. C, C., B, I., W, A., M, E., S, J., G, J. F. & H, W. (1993). Two nuclear signalling pathways for Vitamin-D. Nature, Lond. 361, 657–660. C, V. C. & K, G. (1993). Sequential O-glycosylation of nuclear pore complex protein gp62 in vitro. Eur. J. Cell. Biol. 60, 185–195. C, B. F. (1981). Alpha-fetoprotein: a review. CRC Crit. Rev., Clin. Lab. Sci. 15, 127–185. C, P., K, J., H, M., J, G., H, S., K, C. R., G, P., S, C., M, K. & S, J. (1993). ATP induces a conformational change of the 90-kD heat shock protein (hsp 90). J. biol. Chem. 268, 1901–1907.
111
D, F. C., S, L. C., T, L. P., A, H. & P, W. B. (1991). Localization of the 90-kDa heat shock protein-binding site within the hormone-binding domain of the glucocorticoid receptor by peptide competition. J. biol. Chem. 266, 3482–3490. D, C. V. & L, W. M. F. (1989). Nuclear and nucleolar targeting sequences of c-erbA, c-myb, N-myc, p53, HSP70, and HIV tat proteins. J. biol. Chem. 264, 18019–18023. D, H. F. (1991). In: Chemistry and Biology of a-fetoproteins. Adv. Cancer Res. 56, 253–312. E, R. M. (1988). The steroid and thyroid hormone receptor superfamily. Science 240, 889–895. F, B. M. & S, H. H. (1990). Interactions among a subfamily of nuclear hormone receptors: the regulatory zipper model. Mol. Endocrinol. 4, 1293–1301. F, M., L, C. M., F, J. D., A, H., S, T. J., M, G. C. & G, J. (1992). A ligand-induced conformational change in the estrogen receptor is localized in the steroid binding domain. Biochemistry 31, 5303–5311. G-B, J., H, J. & H, M. N. (1991). Nuclear protein localization. Biochem. Biophys. Acta. 1071, 83–101. G, H. (1992). Control of transcription activation by steroid hormone receptors. FASEB J. 6, 2524–2529. G-M, A., L, H., L, P., S, S., A, M., P-A, M. & M, E. (1989). Mechanisms of nuclear localization of the progesterone receptor: Evidence for interaction of monomers. Cell. 57, 1147–1154. H, M., K¨, S., O, S. & H, T. (1993). Purification of a 60 kD nuclear localization signal binding protein in rat liver nuclear envelopes and characterization of its properties. J. Biochem. 113, 308–313. H-G, M., N, J., T, J. & U, J. (1985). The uptake of AFP by C-1300 mouse neuroblastoma cells. Br. J. Cancer 51, 791–794. H, J. & P, M. G. (1989). Regulation of gene expression by nuclear hormone receptors. Curr. Opin. Cell. Biol. 1, 503–511. H, Z. D., Y, C. R., F, B. M., C, J. & S, H. H. (1989). Characterization of the domain structure of chick c-erbA by deletion mutation: in vitro translation and cell transfection studies. Molec. Endocrinol. 3, 148–158. J, H. I., M, D., M, G. J., B, J. A. & A, T. T. (1989). Estradiol-induced changes in spectral and biologic properties of alpha-fetoprotein. Proc. Int. Soc. Oncodev. Biol. Med. 17, 27. J, H. I., B, J. A. & M, G. J. (1990). Inhibition of estrogen-dependent breast cancer growth by a reaction product of a-fetoprotein and estradiol. Cancer Res. 50, 415–420. LC, E. C., L, H. A., W, P. & L, Y. A. (1993). Identification of binding proteins for nuclear localization signals of the glucocorticoid and thyroid receptors. Endocrinology 132, 1017–1025. L, M. P. (1991). The characterization of a novel, widespread PNA-reactive tumor associated antigen: The alpha-fetoprotein receptor/binding protein. PhD Thesis, The University of Alberta Library, Dept. of Medical Sciences, Edmonton, Alberta, Canada. L, J. D. J., D, L. I. & F, G. R. (1993). NUP2, a novel year nucleoporin, has functional overlap with other proteins of the nuclear pore complex. Molec. Biol. Cell. 4, 209–222. L, G., S, F., W, P., H, J. M., B, J. D. & K, P. J. (1993). Positive and negative modulation of Jun action by thyroid hormone receptor at a unique AP1 site. Molec. Cell. Biol. 13, 3042–3049. M, A. O., D, L. M., M, L. J., L, K., K, J., T, R. & F, N. (1990). Maternal serum alpha fetoprotein complicated by diabetes: Screening Implications. Am. J. Obstet. Gyn. 163, 1209–1216. M, G. J. (1994). Alpha-fetoprotein binding proteins: Implications for transmembrane passage and subcellular localization. Life Sci. 56, 1–9.
112
. .
M, G. J. (1993). An apparent dimerization motif in the third domain of alpha-fetoprotein: Molicular mimicry of the Steroid/Thyroid nuclear receptor superfamily. Bioessays 15, 427–432. M, G. J., S, B. R. & J, H. I. (1989). AFP modification of biologic response in estrogen-sensitive tissues: use of in vivo and in vitro models. In: Biological Activities of Alpha-fetoprotein, Vol. II (Mizejewski, G. J. & Jacobson, H. I., eds) pp. 59–74. Boca Raton, FL: CRC Press. M, G. J., V, M. & J, H. I. (1983). Estradiol-activated a-fetoprotein suppresses the uterotrophic response to estrogens. Proc. natn. Acad. Sci. U.S.A. 80, 2733–2737. M, G. J., K, J. F. & S, R. P. (1990). Separation of the estrogen-activated growth regulatory forms of alphafetoprotein in mouse amniotic fluid. Biol. Reprod. 42, 887–898. M, G. J. (1985). New insights into AFP structure and function: potential biomedical applications. In: Alphafetoprotein and Congenital Disorders (Mizejewski, G. J. & Porter, I. H. eds.) pp. 5–34. Orlando, FL: Academic Press. M, G. J. & W, A. S. (1989). Alpha-fetoprotein can regulate growth in the uterus of the immature and adult ovariectomized mouse. J. Reprod. Fertil. 85, 177–185. M, G. J. & B-B, M. M. (1987). Release of AFP from serine protease inhibitors in serum and tissue cytosols. In: Biological Activities of Alpha-fetoprotein, Vol. II (Mizejewski, G. J. & Jacobson, H. I. eds) pp. 162–179. Boca Rato, FL: CRC Press. M, R., T, T., W, T. G., L, B. M. & L, M. P. (1993). Monoclonal antibodies directed against a widespread oncofetal antigen: the alpha-fetoprotein receptor. Tumor Biol. 14, 116–130. M, H., B, G. & P, D. (1993). Signal sequence region of mitochondrial precursor proteins binds to mitochondrial import receptor. Proc. natn. Acad. Sci. U.S.A. 90, 3358–3362. N, J., V, M. J., G, A. F. & U, J. (1985). Cell type specific receptors for AFP in a mouse T-lymphoma cell line. Proc. natn. Acad. Sci. U.S.A. 82, 3301–3304. N, D. D. & F, D. J. (1990). An N-ethylmaleimide-sensitive cytosolic factor necessary for protein import: Requirement in signal-mediated binding to the nuclear pore. J. Cell. Biol. 100, 547–557. N, S., M, H., Y, H., Y, R. & S, M. (1991). Localization of the estrogen-binding site of a-fetoprotein in the chimeric human-rat proteins. Proc. natn. Acad. Sci. U.S.A. 88, 3102–3105. O, E., H, L. M., & M, A. (1993). Kinetics of glucocorticoid receptor phosphorylation in intact cells. J. biol. Chem. 268, 7779–7784. P, D. & Y, K. R. (1987). Two signals mediate hormone-dependent nuclear localization of the glucocorticoid receptor. EMBO J. 6, 3333–3340. P, D., K, V., C, P. & Y, K. R. (1990). Signal transduction by steroid hormones: nuclear localization is differentially regulated in estrogen and glucocorticoid receptors. Cell. Reg. 1, 291–299. P, W. B., C, M. J., S, L. F. & O, J. K. (1993). The HSP 56 immunophilin component of steroid receptor heterocomplexes: Could this be the elusive nuclear localization signal binding protein? J. Steroid Biochem. molec. Biol. 46, 269–279. R, A., B, G. & W, R. W. (1993). NupL155 is a novel nuclear pore complex protein that contains neither repetitive sequence motifs nor reacts with WGA. J. Cell. Biol. 121, 1–9. R, T. A. (1991). Protein transport across the endoplasmic reticulum membrane: facts, models, mysteries. FASEB J. 5, 2792–2798. R, M., B, W. & G, U. (1991). Protein components of the non-activated glucocorticoid receptor. J. biol. Chem. 266, 24601–24605. R, B. (1989). Nuclear location signal-mediated protein transport. Biochem. Biophys. Acta. 1008, 263–280.
R, E. D., O’D, A. L. & K, R. J. (1991). Protein–protein interactions involving erbA superfamily receptors: through the TRAP door. Molec. Cell. Endocrinol. 78, C83–C88. R, J. E. (1989). Polypeptide chain binding proteins: catalysts of protein folding and related processes in cells. Cell 59, 591–601. R, A. H., S, V., F, M., B, G. & V, J. (1981). Relation of glucose to alphafetoprotein in amniotic fluid. Obs. & Gyn. 57, 310–312. S, E. J., Z, M. & D, D. S. (1983). M, G. J. & J, H. I. Detection and measurement of alpha-fetoprotein in human breast cancer cytosol after treatment with 0.4 M potassium chloride. Cancer Res. 43, 3739–3741. S, E. J. & S, J. R. (1976). Intracellular synthesis of alpha-fetoprotein and fibrinogen without secretion by zajdela rat hepatoma cells. Cancer Res. 36, 3203–3205. S, G. (1993). The protein import machinery of mitochondria. Protein Sci. 2, 141–146. S, G. & G, M. C. (1992). Activation of Kb-specific proteins by estradiol. Proc. natn. Acad. Sci. U.S.A. 89, 10628–10632. S, P. A. (1991). How proteins enter the nucleus. Cell 64, 489–497. S, J. A., S, M., L, M. V., F, F. S. & W, E. M. (1991). Transcriptional activation and nuclear targeting signals of the human androgen receptor. J. biol. Chem. 266, 510–518. S, D. E. & T, D. O. (1993). Steroid receptors and their associated proteins. Molec. Endocrinol. 7, 4–11. S, B. & W, H. J. (1993). The amino-terminal domain of the lamin-B receptor is a nuclear envelope targeting signal. J. Cell. Biol. 120, 1093–1100. S, U. K., R, C. J. & D, M. R. (1987). Heat-induced expression of albumin during early states of rat embryo development. Molec. Cell. Biol. 7, 4599–4602. S, U. K. & R, C. J. (1994). Altered expression of alpha-fetoprotein and albumin in early embryonic rat liver upon heat shock. Biochem. Molec. Biol. Int. 33, 21–28. S, C. M., D’O, P, M. K. & H, J. A. (1990). Primary sequence and heterologous expression of nuclear pore glycoprotein. p. 62. J. Cell. Biol. 110, 1861–1871. S, U. & S, P. (1992). Nucleocytoplasmic traffic of proteins. Eur. J. Cell. Biol. 59, 1–11. S, J. & B, G. (1993). A nuclear pore complex protein that contains zinc finger motifs, binds DNA and faces the nucleoplasm. Cell, 72, 29–38. S, Y., Z, C. Q. Y. & A, E. (1992). Isolation and characterization of a specific alpha-fetoprotein receptor on human monocytes. J. Clin. Invest. 90, 1530–1536. T, Y. S. (1965). Content of embryo-specific alpha-globulin in the blood serum of the human foetus; newborn, and adult man in primary cancer of the liver. Vop. Khim. SSSR 11, 20–24. T, T. B. (1977). Structure and Function of alpha-fetoprotein. A. Rev. Med. 28, 453–465. T, T. C. (1993). New Model for 70 kDa heat-shock proteins’ potential mechanism of function. FEBS Lett. 323, 1–3. T, P., P, A., B, M., J, T., V, P. & Y, T. (1993). Nuclear progesterone receptor is mainly heat shock protein 90-free in vivo. Proc. natn. Acad. Sci. U.S.A. 90, 5848–5852. U, J., N, J. & L, J. (1986). Alpha-fetoprotein mediated transfer of arachidonic acid into cultural cloned cells derived from a rat rhabdomyosarcoma. J. biol. Chem. 262, 3579–3585. U, J., F-C, C., V, M. J., P, A. & G, M. (1984). Incorporation of alpha-fetoprotein by the MCF-7 human breast cancer cell line. Tumor Biol. 5, 41–51. W, W. & M, E. (1991). Superfamily of steroid nuclear receptors: positive and negative regulators of gene expression. FASEB J. 5, 2243–2249. W, S. R., R, M. P. & B, G. (1992). A new family of yeast nuclear pore complex proteins. J. Cell. Biol. 119, 705–723.
/ W, S. T. & G, S. (1993). Signal transduction and nuclear targeting: regulation of transcription factor activity by subcellular localization. J. Cell. Sci. 104, 949–955. W, F. G., N, M., & S, C. (1992). Candidate protonocogene bcl-3 encodes a subunit-specific inhibitor of transcription factor NF-kB. Nature, Lond. 358, 597–599. Y, L., K, P. & L, R. E. (1989). Identification of four nuclear transport signal-binding proteins that interact with diverse transport signals. Molec. Cell. Biol. 9, 3028–3036.
113
Y, F. & O, M. (1992). The gene for the rat heat-shock cognate, hsc 70, can suppress oncogene-mediated transformation. Cell Growth Diff. 3, 803–809. Y, T., B, M. T., B, M., G, H. & C, P. (1992). Cooperation of proto-signals for nuclear accumulation of estrogen and progesterone receptors. EMBO J. 11, 3681–3694. Z, K. V., A, F. & F, E. (1991). A hydrophobic protein sequence can override a nuclear localization signal independently of protein context. Molec. Cell. Biol. 11, 5137–5146.
Alpha-fetoprotein Signal Sequences: A Proposed Mechanism for Subcellular Localization and Organelle Targeting G. J. M Laboratory of Human Genetics, Wadsworth Center for Lab & Research, NYS Dept. of Health, Albany, New York 12201, U.S.A. (Received on 26 October 1994, Accepted in revised form on 17 April 1995)
Alpha-fetoprotein (AFP) is an oncofetal protein, classified in an ‘‘albuminoid’’ superfamily (with albumin and Vitamin-D binding (Gc) protein) comprising molecules with three characteristic globular domains. The cellular uptake and internalization of AFP and its subcellular compartmentalization has previously been reported in a multitude of cell types. Studies have also emerged which have detected and characterized binding proteins complexed to AFP in various cell membranes and intracellular compartments. However, the literature is devoid of any attempts to relate these binding proteins to possible intracellular trafficking interactions of AFP. Recombinant DNA mutation/deletion technology has provided a means to pinpoint the amino acid sequence location of organelle localization signals on various transcription factors and/or receptors. Several subdomain regions on AFP have been reported to mimic heptad dimerization regions of the steroid/thyroid nuclear receptors. In light of these transcription factor-like docking motifs reported for AFP, the present report purposes various subdomain regions which might constitute basic amino acid sequences resembling recognition signals for binding (dimerizing) proteins. AFP appears to possess multiple prototypic amino acid sequence cassettes on each domain which consist of (i) classical short, compact sequences found on both steroid and thyroid receptors; (ii) proto-signals resembling a steroid receptor type; and (iii) degenerative signal sequence similar to the thyroid/retinoic acid receptor type. The concepts identifying binding or escort proteins for AFP together with organelle signal sequences on AFP have been reconciled in a composite hypothesis to formulate a mechanism which could account for some of the growth-regulating properties described for AFP during the last decade. 7 1995 Academic Press Limited
During the last 20 years, a plethora of studies has emerged implicating the existence of AFP in a form complexed with other proteins or peptides (see review, Mizejewski, 1994). This bound form of AFP is found in both sera and tissue extracts and is dissociable by the use of high salt (0.4 M KCl) or serine protease inhibitors (Sarcione et al., 1983; Mizejewski & Brown-Biddle, 1989). To date, the physiological significance of this bound-form of AFP has eluded clarification. Earlier studies of AFP function documented that this fetal protein displayed serum binding/transport properties regarding fatty acids, steroids, retinoic acid, heavy metals, and various drugs (Tomasi, 1977). More recent studies have demonstrated that AFP is capable of both oncogenic and ontogenetic growth regulation,
Introduction During ontogenic development in mammals, products termed oncofetal or phase-specific proteins are produced at various stages of embryonic, fetal and neoplastic growth (Tatrinov, 1965; Abelev, 1971). One of the most widely studied onofetal proteins to date is alpha-fetoprotein (AFP), an alph-1glycoprotein comprised of a single-chain 70 kDa polypeptide containing 3–5% carbohydrate (Mizejewski, 1985; Deutsch, 1991). AFP is also produced and secreted in patients with hepatocellular carcinomas, teratoblastomas, hepatitis, and cirrhosis (Crandall, 1981). Thus, the presence of circulating AFP in serum levels renders it a useful marker for fetal distress, tumor growth and hepatic dysfunction. 0022–5193/95/170103+11 $12.00/0
103
7 1995 Academic Press Limited
104
. .
F. 1.—caption opposite.
/ and that such modulation might be attributed to transcriptional enhancement and/or suppression activities (Mizejewski & Warner, 1989; Mizejewski et al., 1990). It has also been documented that AFP displays both cellular uptake and transmembrane passage via specific cell surface receptors (Laderoute, 1991; Moro et al., 1993) and cytoplasmic binding proteins culminating in either receptosome or lysosomal pathways (see review, Mizejewski, 1994; Suzuki et al., 1992). It may be hypothesized that AFP, once endocytosed, might be capable of diverse subcellular localization by means of amino acid signal-like sequences which could affect organelle targeting. In the present treatise, the reports involving the uptake of AFP, the bound forms of AFP, findings of presumptive amino acid signal sequences, and the observations of growth regulatory properties of this oncofetal protein have been merged to propose a mechanism implicating these AFP activities with intra-cellular localization and organelle targeting. Intracellular Organelle Targeting During the period in which AFP-binding proteins were observed and reported, the functional domains of the steroid/thyroid nuclear receptor superfamily members were being characterized using recombinant DNA deletion/mutation technology (Evans, 1988; Ham & Parker, 1989). This superfamily of nuclear receptors are known to be inducible transcriptional regulators which bind steroid/thyroid/vitamin ligands and recognize hormone response elements of target genes to enhance or repress transcription (Wahli & Martinez, 1991). The functional domains of these receptors has been assigned the letters A to F in which C is the DNA binding domain and E is the ligand binding domain (Forman & Samuels, 1990). The D domain has not been as extensively characterized but is thought to contain a major nuclear localization signal (NLS) which serves as a targeting site to transport the receptor into the nucleus (Gronemeyer, 1992). However, other NLS sites have also been identified in the C and E domains and appeared to
105
function in concert or in a cooperative fashion to enact organelle translocation (Ylikomi et al., 1992; Dang et al., 1989). Although a consensus sequence has not yet been ascertained, the targeting signals are thought to consist of three types: (1) a concise, compact stretch of basic amino acids (usually 3–7 residues); (2) two or more weaker and less concise proto-signals containing clusters of basic amino acids interspersed with neutral amino acids; and (3) multiple degenerative sequences which are extended in length, noncontiguous, scattered residues of basic amino acids (Roberts, 1989; Garcia-Bustos et al., 1989). The compact amino acid sequence signals are frequently flanked by either glycine, proline or acidic amino acids (Silver, 1991). It has been proposed that the presence of neutral amino acids flanking the NLS provide insulating boundaries for the signal site or serve other modulating functions (Stochaj & Silver, 1992). Proteins bearing such signal amino acid sequences are known to bind a cytoplasmic signal recognition receptor (55–75 kDa binding proteins) which then escorts the signal bearing protein to various target organelles (i.e. nuclear pore complex, outer mitochondrial pore, etc.) (Adam et al., 1989; Schatz, 1993). Figure 1 represents a compilation of the various NLS’s found in various members of the steroid/ thyroid/vitamin receptor superfamily (Picard et al., 1990; Dang et al., 1989; Guiochon-Mantel et al., 1989); these were then compared to presumptive signal amino acid cassette sites proposed for the AFP molecule. It became immediately apparent that the AFP molecule itself displayed intrinsic prototypic signal-like sequence homologies among its own three domains. There further appeared to be multiples of the different signal types on all three domains of AFP. First, four compact type of signal-like cassettes present on all three domains of AFP appeared to resemble those of the thyroid/retinoic acid receptors more than the steroid receptor family members, although both share similarities.
F. 1. Amino acid sequence alignment of the various organelle localization signal cassette sites proposed for rodent and human alpha-fetoprotein versus the established sites on the steroid/thyroid related receptors. The amino acid (AA) sequences of the various classes of organelle targeting signals are listed for both mouse (M) and human (H) alpha-fetoprotein and the nuclear receptor superfamily members: (a) Class I localization signals are compact, concise localization sites consisting of three to seven basic residues shown with surrounding AAs. (b) Class-II type localization proto-signal sites comprised two or more weaker and less concise acidic AA stretches in a bipartite fashion. (c) Class-III type localization degenerative sequences are extended in length, non-contiguous, with scattered residues of basic AAs. AFP, alpha-fetoprotein, AR, androgen receptor; ER estrogen receptor; GR, glucocorticoid receptor; MR, mineralcorticoid receptor; PR, progesterone receptor; RAR, retinoic acid receptor; RXR, retinoic acid-X-receptor; VDR, Vitamin D receptor. All receptors denote the human species; T3R represents the B-isoform, while RAR and RXR, the a-isoforms.
106
. .
Second, two signal sites within the steroid binding domain bore resemblance to comparable bipartite proto-signal sites reported for the steroid nuclear receptor members (LaCasse et al., 1993). Third, two of the localization sites appeared to comprise part of a more degenerative signal sequence (Horowitz et al., 1989), one site of which lies on the second domain of AFP. Among steroid receptor superfamily members, cytoplasmic receptor residence has been noted for the glucocorticoid, mineralcorticoid, androgen and the vitamin-D receptors (Simental et al., 1991; Carlberg et al., 1993). For purposes of comparison, the glucocorticoid receptor provides a good cytoplasmic example since the estrogen and progesterone receptors are thought to reside principally in the nucleus, while the glucocorticoid receptor has been localized in nonnuclear cytoplasmic fractions (Guiochon-Mantel et al., 1989). The glucocorticoid receptor contained a major NLS positioned at the carboxy terminal end of the second zinc finger in the D-domain (termed NL1); an NL2 region is located in the hormone (ligand) binding region of the E-domain (Ylikomi et al., 1992; Picard et al., 1990). The NL1 would correspond to the D-domain of most steroid receptors and might be exemplified on AFP as amino acid sequences 339–354 in which a series of basic compact, concise amino acids reside at 341–345. The NL2 region of the glucocorticoid receptor contains two subregions that require hormone-binding for activation, one located toward the amino-end of the ligand binding domain and the other at the carboxy-terminal last third of the domain. These two NLS regions on the glucocorticoid receptor might be analogous to the proposed protosignal amino acid sequences 416–431 and 465–478 on AFP. In the glucocorticoid receptor, these regions of the ligand binding domain required hormone for activation; thus, it may be more than coincidence that AFP amino acid sequences 416–431 and 465–478 lie at the beginning and end of the estrogen binding subdomain reported for rodent AFP (Nishi et al., 1991). The latter proto-signal which appeared to be present at AFP amino acids 465–478 lies directly in a region crucial to steroid (estrogen) binding regulation in the third domain of AFP (Nishi et al., 1991). These two regions on AFP could serve as potential proto-signals for organelle (nuclear) accumulation as reported for the glucocorticoid, estrogen and progesterone receptors (LaCasse et al., 1993; Picard et al., 1987). A concise signal in the third domain of human AFP might be located within amino acids 537–562 (532–545 in Fig. 1) which lie in a strongly hydrophobic region of leucine zipper-like heptad repeats resembling
a stretch of 100 amino acids comprising a large putative dimerization subdomain found in the nuclear receptor superfamily (Mizejewski, 1993). It has been demonstrated that a strongly hydrophobic carboxy terminus sequence can override a NLS independently of the amino acid context of the signal-bearing protein in the cytoplasm (Zee et al., 1991). Upon binding E2, the rat uterine estrogen receptor has been reported to undergo a conformational change exemplified by a dramatic decrease in surface hydrophobicity localized toward the carboxy-terminal side of the steroid binding domain (Fritsch et al., 1992). In comparison, AFP is known also to undergo a conformational change in the presence of high E2 concentrations, as evidenced by a change in the UV difference spectrum (Jacobson et al., 1989). This time-dependent absorbance maximum of AFP displayed a high molar extinction coefficient suggesting a positional change in intrinsic aromatic amino acids. A conformational change on AFP, as a result of E2 exposure results in a dramatic change in carboxy terminus hydrophobicity, which could have relieved a hydrophobic override in the signal site and exposed the amino acid localization sequence for subsequent organelle targeting. Similar intramolecular masking effects has been reported for several other proteins including NF-kb, c-Rel, dorsal and the glucocorticoid receptor (Whiteside & Goodbowen, 1993), and could apply to AFP (Table 1) in light of recent studies demonstrating AFP regulation of estrogen-induced uterine and tumor growth (Mizejewski et al., 1983; Jacobson et al., 1990; Mizejewski et al., 1990). Finally, molecular masking is also thought to occur via small ligand molecules (peptides, organic molecules, protease inhibitors/substrates, etc.) binding at or overlapping onto organelle cassette signal sites (Roberts, 1989; Silver, 1991). Studies with synthetic peptide fragments bound to the LBD as co-transporters have confirmed such findings in the steroid receptor superfamily (Dalmon et al., 1991). Unlike the glucocorticoid receptor, estrogen and progesterone receptors are known to reside largely as nucleoplasmic receptors and display a different array of NLS’s (Picard et al., 1990). The estrogen and progesterone receptors contain multiple proto-signals (pNLS) utilized for nuclear targeting (none of which suffices on its own) which cooperate or act in concert as a result of a ligand-induced conformational assemblage. The estrogen receptor contains an estrogen-induced pNLS in addition to three other basic amino acid-rich proto-signals which cooperate following exposure to estrogen (Ylikomi et al., 1992). Thus, AFP might mimic the estrogen receptor in displaying multiple weaker signal-like sites (amino
/
107
T 1 Various cellular transcription factor and/or DNA binding proteins and their respective inhibitors DNA binding protein/complex 1. Steroid/thyroid receptors (complex) 2. NF-kB complex (rel-Family) 3. AP-1, activator protein-1 4. MyoD
Related or component subunits
Type of oligomer formation
Apparent subcellular residence
ER, PR GR, MR AR, RAR RXR, TR, RAR p50 p65 p50 p52 C-JUN C-FOS C-myc E12 E47
Monomers Monomers HomoHeterodimers Heterodimers
Cytoplasm and nuclear
HSP-90 HSP-70 HSP-59
Activation, inhibition
Cytoplasm
Heterodimers
Nucleus (cytoplasm) Cytoplasm
lkB (43 kDa) BcL-3 (46–56 kDa) 1P-1 (37 kDa) 1D (43 kDa)
Activation, inhibition Activation (enhancement) Enhancement, inhibition Enhancement, inhibition
HomoHeterodimers HomoHeterodimers
Cytoplasm
Family of inhibitors
Transcriptional regulation
Reference Smith, 1993 Cadepond, 1991 Rexin, 1991 Dalmon, 1991 Shyamala, 1992 Gilmore, 1993 Wulczyn, 1992 Lopez, 1993 Avwerx, 1991 Lasser, 1989 Davis, 1990
Such factors could serve as prime candidates as the cytoplasmic alpha-fetoprotein binding proteins. These all represent cellular immediate-early response gene products, many of which utilize protein kinase C (PKC) signal transduction pathways following cell stimulation by serum factors and/or phorbol esters (tPA, 12-0-tetradeconyl-phorbol-13-acetate). The present report proposes that AFP binding to the transcription factor (DNA-binding protein) would result in growth inhibition, while AFP complexing to the inhibitor would cause growth enhancement. Abbreviations used: ER, estrogen receptor; erbA, putative thyroid hormone receptor proto-oncogene products; MR, mineralcorticoid response element on DNA; RAR, retinoic acid receptor; RXR, retinoic-X-receptor, T3R, thyroid hormone (triiodothyronine) receptor; VDR, vitamin D receptor.
acids 416–431, 465–478). A fourth suspected NLS of the estrogen receptor, although more degenerative, could exist toward the amino terminal side of the DNA binding domain. Such discontinuous motifs are known to exist in other superfamily members (such as retinoic acid, thyroid, retinoic-X and mineralocorticoid receptors, etc.) (La Casse et al., 1993; Horowitz et al., 1989), and AFP may indeed have such a weak NLS-like site at amino acid sequences 223–237. Other degenerative (weak) presumptive sites on the first domain of AFP at amino acids 80–94, 102–116 and 142–156 could also serve as presumptive signals for other organelle targeting. Frequently, the signals toward the amino-terminal portion of other proteins (i.e. prepeptide signal piece) are associated with targeting to organelles such as the mitochondria, Golgi, ribosomes, and endoplasmic reticulum (Roise & Schatz, 1988; Rapaport, 1991), although a lamin receptor contains an amino terminal NLS site (Soullam & Worman, 1993). Thus, these proposed amino-terminal localization-like cassettes on AFP might represent mitochondrial, microsomal or EPRrelated signals (see Class 1, Fig. 1) in contrast to NLS’s. It may be no coincidence that one of the proposed signal cassette sites on AFP (amino acids 532–545) is associated with a presumptive hetero-dimerization region which has previously been purposed for this fetal protein. Since the nuclear targeting escort proteins usually recognize a targeting signal, it would seem reasonable to find (as in AFP) the dimerization sites located in the near vicinity, either
flanking the signal cassette or inclusive with it. This observation would suggest a direct association between the presence of the signal cassette and the location of the dimerization interface. It would be conceivable that NLS sites on proteins also function as dimerization recognition and/or docking sites (Smith & Toft, 1993). Pratt et al. (1993) have proposed that the arginine/lysine-rich sequences comprising the signal site would bind to the escort protein displaying signal docking glutamic/aspartic acid-rich sequences. In this regard, it has already been demonstrated that the signal sequence site on precursor proteins bind to the organelle import receptors intended for mitochondrial targeting (Murakami et al., 1993).
Binding Proteins with NLS Recognition It would seem that AFP may somehow be capable of interacting with nucleocytoplasmic trafficking or organelle targeting since it appeared to contain multiple presumptive signal sites (Fig. 1). Cytoplasmic proteins that specifically recognize and bind organelle signal-proteins have been reported to serve as escort receptors for nuclear import (Adam & Gerace, 1991; Haino et al., 1993). Interaction of NLS-containing receptors with the signal-bearing protein was the initial step in mediated nuclear import. The cytoplasmic binding proteins are reported to range in size from
108
. .
56–76 kDa or greater while the nuclear poreassociated proteins appear larger, approximating 140–185 kDa (Rosen et al., 1991; Sukeqawa & Blabel, 1993). The initial binding of the signal protein to the nuclear membrane is associated with high affinity binding is saturable, dependent upon the presence of cytosol, and is sensitive to inactivation by N-ethylmaleimide (Newmeyer & Forbes, 1990). A subsequent step was an NLS-dependent binding to the cytoplasmic side of the nuclear pore complex (NSC) based on in vivo and in vitro studies using colloidal gold probes (Adam et al., 1989; Adam & Gerace, 1991). Thus, the NPC appeared to serve as a morphological connection between the nuclear and cytoplasmic compartments and could potentially exert a powerful influence on controlling gene activity via transport of regulatory components for the selective interaction with certain transcribed genes (i.e. gene gating; see below). A family of cytoplasmic glycoproteins termed nucleoporins bearing O-linked N-acetylglucosamines have been identified in eukaryotes of which rat nucleoporin p62 is a member (Starr et al., 1990; Loeb et al., 1993). This karyophilic protein consists of three domains of which the carboxy-terminal third domain displays heptad repeats comprising a dimerization domain. It is noteworthy that Laderoute (1991) and Moro et al. (1993), using monoclonal antibodies, identified AFP-binding protein doublet moieties at 62 and 67 kDa in which the binding proteins contained O-linked glycosylation sites as found in other systems (Cordes et al., 1993). Similarly, identification of binding proteins for the NLS site of the glucocorticoid and thyroid-hormone (triiodothyronine) receptors have been reported in lymphoma cytosols as 60 and 76 kDa kinase-related polypeptides (LaCasse et al., 1993). It is tempting to speculate that the binding of at least some of the cytosolic and saturable membrane-associated protein binding to AFP could actually be a class of NLS recognition escort proteins. Alternatively, AFP itself might serve as a NLS recognition binding protein (Table 1). It is of particular interest that non-liganded progesterone receptor monomer was shown to dimerize with the liganded monomer, forming oligomers (dimers) that are transported into and out of the nucleus (Guiochon-Mantel et al., 1989). Thus, the NLS receptors themselves might function to shuttle receptors and nucleoproteins between the cytoplasm and the nucleus in a bidirectional fashion. One could speculate that liganded AFP could have interacted with non-liganded AFP to help explain the results of some previous experiments where E2-treated AFP was capable of growth regulation in normal and
neoplastic reproductive tissues (Jacobson et al., 1990; Mizejewski et al., 1990). It is further conceivable that AFP, when exposed to a high concentration of ligands, converts AFP into a signal sequence binding protein, which could recruit non-liganded proteins (i.e. AFP?) transcription factors, or steroid receptors (Table 1) as its dimer partner for cytoplasmic translocation toward the organelle surface (nuclear) (Panels II & III, Fig. 2) or inhibition of that transport. Transorganelle (transnuclear) passage involves interaction of the signal site bearing protein with a putative transporter protein assembly situated in or about the central channel of the NPC (Loeb et al., 1993; Cordes et al., 1993). Translocation of the import protein through the central channel of the NPC requires ATP phosphorylation and is accompanied by size-selective gating at the channel pore (Wente et al., 1992; Radu et al., 1993). This phosphorylation step is inhibited by reduced temperatures and by wheat germ agglutinin lectin. Pore complex proteins of 153 kDa (180 kDa in SDS–PAGE), which have been cloned and sequenced, contained zinc fingers, bound DNA, and faced the nucleoplasmic interface (Sukegawa & Blobel, 1993). It is thought that the 153 kDa could be a DNA binding subunit of the NPC that could link the complex to cognate DNA sequences thereby gating transducable genes to the pore complex. It is interesting that Yamasaki, et al. (1989) also identified a 140 kD NLS binding protein that associated with rat liver nuclei. As previously reported, an AFP-binding protein of 185 kDa (SDS) has been detected in the solubilized membrane fraction from breast cancer cells (Laderoute, 1991). Reaction of monoclonal antibodies to the AFP-binding protein further resulted in phosphorylation of this protein involving ATP release. This protein could be likened to the 153 (185) kDa protein requiring ATP phosphorylation as described above. Is AFP a Heat Shock Protein? One of the most interesting properties of HSP-70 is its ability to form complexes with a variety of viral oncogene products (Yehiely & Oren, 1992). In fact, it has been proposed that heat shock proteins function as cross-linking agents, especially cytoskeletal (actin) elements, to facilitate physiological cascade phenomenon (Tsang, 1993). In a similar fashion, the c-erbA dimerization sites proposed for AFP in a previous study has been advanced in attempting to explain the regulation of growth by AFP as reported in previous investigations (Mizewjewski, 1993). It is conceivable that AFP could function as a heat shock cognate-like
/
109
F. 2. The uptake, transmembrane passage, and metabolic fate of liganded and free AFP are depicted in an epithelial cell model depicting four possible metabolic pathways for AFP. The cell is subdivided into four panels (I–IV). Panel I depicts both liganded and non-liganded AFP and Albumin (ALB) endocytosis which has been documented in the literature. Panel II proposes that AFP be liganded displaying partial signal site exposure and conformational alteration leading to an endocytotic pathway involving endoplasmic reticulum (EPR) entry toward the nuclear pore. Panel III displays a similar vesicular uptake of AFP, except that AFP is released from the endosome directly into the cytoplasm. Panel IV portrays an endocytotic liganded pathway in which AFP is also released directly into the cytoplasm or could apply to AFP synthesized but not secreted from the cell. CMR, cell membrane receptor; DBP, DNA-binding protein; EL, endolyzozome, EPR, endoplasmic reticulum; ER, estrogen receptor; EV, endosomal vesical; GC, Golgi complex; HRE, hormone response element; HSP, heat shock protein; N, nucleus; NBP, nuclear binding protein; RP, receptor-binding protein.
protein or an accessory protein and bind to the nuclear receptor complex at their heat shock protein designated sites or perhaps dimerize or compete with the heat shock protein itself. It has been reported that the HSP-90 protein appears to cap the steroid receptor DNA-binding region, preventing it from binding to the DNA hormone response element thereby stabilizing the receptor in the cytoplasm (Cadepond et al., 1991). Dimerization of heat shock protein to AFP could render the steroid nuclear receptor more susceptible to degradation and possibly disrupt kinase activities. Alternatively, the heat shock protein and/or AFP could either mask a signal region or interfere with the localization recognition site which is linked to receptor activation since
phosphorylation was found coincident with HSP-90 dissociation from the 9S glucocorticoid receptor complex following a conformational change (Orti et al., 1993; Csermely et al., 1993). It has been reported that albumin synthesis could be induced by heat shock stimulation in fetal rats (Srinivas et al., 1987); however, heat shock stimulation of AFP resulted only in an unstable, degraded AFP mRNA while circulating AFP was unaffected (Srinivas & Revathi, 1994). Since albumin has recently been shown to bind the ATP molecule (Bauer et al., 1992), the binding of ATP to AFP is also possible based on crystal structure molecular similarities. Although, AFP has not been reported to exhibit amino acid sequence homology with the heat shock family of proteins, it is
. .
110
evident that AFP competition, interaction or dimerization with the heat shock proteins have yet to be ruled out. To date, present data would argue against AFP as a heat shock protein if elevated temperature (heat) is used as the criteria. However, glucose-shocked AFP has yet to be studied since glucose levels are known to correlate with circulating AFP levels in the fetus; pregnant women with insulin-dependent diabetes are also known to have lower AFP serum levels than non-affected pregnant women (Martin et al., 1990; Rule et al., 1981). If it were possible to link a heat shock protein-like polypeptide binding function to AFP, then a myriad of additional molecular chaperone roles could be implied. One or more of these functional roles might involve polypeptide chain folding, phosphorylation, maintenance of inactive state for transcription factors, nuclear translocation signalling, cross-talk cell signalling, induction of conformational change (as in receptor complex dissociation), transmembrane passage, regulation of proteolytic degradation of activated receptors, and binding to the heavy chain of IgG (Rothman, 1989). It is noteworthy that AFP has been detected in a form complexed to both immunoglobulin G and M and to serine protease substrates/inhibitors (Mizejewski & Brown-Biddle, 1987). Thus, it is conceivable that AFP could be a heat shock protein-like prototype of the adult stress/growth proteins expressed during the embryonic and fetal life of the organism in which dimerizing to polypeptides may be one of the colligative properties of this protein. AFP Metabolic Pathway Models The above hypothesis stated that AFP might display potential multiple AA sequences which resemble organelle targeting signal sites reminiscent of steroid/ thyroid nuclear receptors. Such sequences could also provide recognition sites to attract cytoplasmic receptor and/or transcription factors for binding or dimerization. Following transmembrane passage of AFP, which has been well documented (Uriel, 1984; Naval et al., 1985), four cell model pathways may be postulated for the metabolic fate of this fetal protein depending on its conformational state (Fig. 2). The first pathway (panel I, Fig. 2) involves a welldocumented shuttle system for endocytosed AFP free or liganded to fatty acids and/or steroids which permits ligand release and recyclization of AFP to the extracellular compartment via the Golgi complex (Uriel et al., 1986; Mizejewski et al., 1989). In a second pathway (panel II, Fig. 2), the cellular uptake of ‘‘partial’’ conformationally altered AFP (incomplete
signal unveiling following exposure to excessive ligand concentrations) might involve receptosome transport to the perinuclear space of the endoplasmic reticulum for possible protein-to-protein interaction. AFP has been histochemically localized within the perinuclear spaces (Hajeri-Gemond et al., 1985); such regions are known to be continuous with channels of the endoplasmic reticulum. A third pathway, involving ligand-induced ‘‘complete’’ conformationally-altered AFP, could involve endosomal release of AFP directly into the cytoplasm (Panel III, Fig. 2). The endosomal release could be related to the dissolving of clathrin coatings reminescent of heat shock protein functions. Conformational transformation could expose full signal recognition sequences on AFP and induce a subsequent binding to cytoplasmic signal recognition escort proteins for import to the nucleus via the nuclear pore in direct contact with cytoplasm. Once within the nucleus, AFP might either dimerize with various members of the steroid/ thyroid receptor superfamily to form inactive heterodimers, bind to repressor/ inhibitory factor targeted for transcription enhancement, or bind nuclear cofactors (i.e. API, RXR, NF-kb, FOS/JUN; Table 1) in transit for transcriptional regulation (Lopez et al., 1993; Whiteside et al., 1993). As a fourth cell model pathway, one cannot rule out the possibility that AFP could bind and retain karyophilic transcription factors and/or their inhibitors in the cytoplasm, thus preventing their import into the nucleus (Panel IV, Fig. 2). This could also apply to AFP synthesized in a cell but not secreted (Sarcione et al., 1976). Thus, by modifying the activities of receptor-associated factors, AFP might function to inhibit translocation before signal (transcription) activation could occur (Panel IV, Table 1). NF-kb nuclear transcription factor, which is found in both lymphoid and uterine cells, is related to the c-rel oncogene and provides a prime example of such DNA-binding factors (Wulcyzn et al., 1992). NF-kb, a 50/65 kDa hetero-dimer, is normally retained in the cytoplasm by a 36 kDa inhibitor termed I-kb and is released upon phosphorylation and/or estrogen stimulation (Shyamela & Guit, 1992). In this regard, it is of great interest that AFP binding proteins of 62–67 kDa mass have been reported (Laderoute, 1991; Moro et al., 1993). Another example, the glucocorticoid receptor, is retained in the cytoplasm complexed to HSP-90 (Rexin et al., 1991). Competing off the heat shock protein from the receptor complex could also result in glucocorticoid receptor confinement to the cytoplasm. In both instances, the protein bound in a complex is unable to bind DNA since their NLS’s are concealed in both the former and latter examples, preventing
/ nuclear import. It is also possible that AFP could bind the cytoplasmic inhibitors of the DNA binding proteins (i.e. IkB or IP-1), allowing subsequent translocation of the nuclear factor to occur (see Fig. 2). Such a mechanism could provide the rationale to account for the growth enhancement previously attributed to AFP. In contrast to the AFP growth inhibition described in the above paragraph, this growth enhancement could occur with AFP whose signal sequences are concealed due to insufficient (incomplete) conformational exposure of the various signal cassettes, the proto-NLS, or the degenerative signal sites (Panel IV, Fig. 2). In Panels III and IV of Fig. 2, AFP need only to reside in the cytoplasm to produce its effect on growth. Concluding Remarks Based on this NLS-binding protein concept together with binding proteins described in previous reports, AFP could serve to either inhibit or enhance gene regulation at the transcriptional level, as previous experimental findings would seem to indicate (Mizejewski et al., 1990; Jacobson et al., 1990). The volume of evidence in the literature supporting AFP as a factor in growth regulation can no longer be ignored. It is hoped that the present report will both arouse interest and provide a working hypothesis to help explain the growth-related properties of AFP to the biomedical community.
REFERENCES A, G. I. (1971). Alpha-fetoprotein in association with malignant tumors. Adv. Cancer Res. 14, 295–357. A, S. A. & G, L. (1991). Cytosolic proteins that specifically bind nuclear location signals are receptors for nuclear import. Cell 66, 837–847. A, S. A., L, T. J., M, M. A. & G, L. (1989). Identification of specific proteins for nuclear location sequence. Nature, Lond. 337, 276–279. B, M., B, J. & T, W. E. (1992). ATP binding to bovine serum albumin. FEBS Lett. 313, 288–290. C, F., S-G, S-M, I., J, H, S. M., G V., E, R. M. & B, E. E. (1991). Heat shock protein-90 as a critical factor in maintaining glucocorticoid receptor in a non-functional state. J. biol. Chem. 266, 5834–5841. C, C., B, I., W, A., M, E., S, J., G, J. F. & H, W. (1993). Two nuclear signalling pathways for Vitamin-D. Nature, Lond. 361, 657–660. C, V. C. & K, G. (1993). Sequential O-glycosylation of nuclear pore complex protein gp62 in vitro. Eur. J. Cell. Biol. 60, 185–195. C, B. F. (1981). Alpha-fetoprotein: a review. CRC Crit. Rev., Clin. Lab. Sci. 15, 127–185. C, P., K, J., H, M., J, G., H, S., K, C. R., G, P., S, C., M, K. & S, J. (1993). ATP induces a conformational change of the 90-kD heat shock protein (hsp 90). J. biol. Chem. 268, 1901–1907.
111
D, F. C., S, L. C., T, L. P., A, H. & P, W. B. (1991). Localization of the 90-kDa heat shock protein-binding site within the hormone-binding domain of the glucocorticoid receptor by peptide competition. J. biol. Chem. 266, 3482–3490. D, C. V. & L, W. M. F. (1989). Nuclear and nucleolar targeting sequences of c-erbA, c-myb, N-myc, p53, HSP70, and HIV tat proteins. J. biol. Chem. 264, 18019–18023. D, H. F. (1991). In: Chemistry and Biology of a-fetoproteins. Adv. Cancer Res. 56, 253–312. E, R. M. (1988). The steroid and thyroid hormone receptor superfamily. Science 240, 889–895. F, B. M. & S, H. H. (1990). Interactions among a subfamily of nuclear hormone receptors: the regulatory zipper model. Mol. Endocrinol. 4, 1293–1301. F, M., L, C. M., F, J. D., A, H., S, T. J., M, G. C. & G, J. (1992). A ligand-induced conformational change in the estrogen receptor is localized in the steroid binding domain. Biochemistry 31, 5303–5311. G-B, J., H, J. & H, M. N. (1991). Nuclear protein localization. Biochem. Biophys. Acta. 1071, 83–101. G, H. (1992). Control of transcription activation by steroid hormone receptors. FASEB J. 6, 2524–2529. G-M, A., L, H., L, P., S, S., A, M., P-A, M. & M, E. (1989). Mechanisms of nuclear localization of the progesterone receptor: Evidence for interaction of monomers. Cell. 57, 1147–1154. H, M., K¨, S., O, S. & H, T. (1993). Purification of a 60 kD nuclear localization signal binding protein in rat liver nuclear envelopes and characterization of its properties. J. Biochem. 113, 308–313. H-G, M., N, J., T, J. & U, J. (1985). The uptake of AFP by C-1300 mouse neuroblastoma cells. Br. J. Cancer 51, 791–794. H, J. & P, M. G. (1989). Regulation of gene expression by nuclear hormone receptors. Curr. Opin. Cell. Biol. 1, 503–511. H, Z. D., Y, C. R., F, B. M., C, J. & S, H. H. (1989). Characterization of the domain structure of chick c-erbA by deletion mutation: in vitro translation and cell transfection studies. Molec. Endocrinol. 3, 148–158. J, H. I., M, D., M, G. J., B, J. A. & A, T. T. (1989). Estradiol-induced changes in spectral and biologic properties of alpha-fetoprotein. Proc. Int. Soc. Oncodev. Biol. Med. 17, 27. J, H. I., B, J. A. & M, G. J. (1990). Inhibition of estrogen-dependent breast cancer growth by a reaction product of a-fetoprotein and estradiol. Cancer Res. 50, 415–420. LC, E. C., L, H. A., W, P. & L, Y. A. (1993). Identification of binding proteins for nuclear localization signals of the glucocorticoid and thyroid receptors. Endocrinology 132, 1017–1025. L, M. P. (1991). The characterization of a novel, widespread PNA-reactive tumor associated antigen: The alpha-fetoprotein receptor/binding protein. PhD Thesis, The University of Alberta Library, Dept. of Medical Sciences, Edmonton, Alberta, Canada. L, J. D. J., D, L. I. & F, G. R. (1993). NUP2, a novel year nucleoporin, has functional overlap with other proteins of the nuclear pore complex. Molec. Biol. Cell. 4, 209–222. L, G., S, F., W, P., H, J. M., B, J. D. & K, P. J. (1993). Positive and negative modulation of Jun action by thyroid hormone receptor at a unique AP1 site. Molec. Cell. Biol. 13, 3042–3049. M, A. O., D, L. M., M, L. J., L, K., K, J., T, R. & F, N. (1990). Maternal serum alpha fetoprotein complicated by diabetes: Screening Implications. Am. J. Obstet. Gyn. 163, 1209–1216. M, G. J. (1994). Alpha-fetoprotein binding proteins: Implications for transmembrane passage and subcellular localization. Life Sci. 56, 1–9.
112
. .
M, G. J. (1993). An apparent dimerization motif in the third domain of alpha-fetoprotein: Molicular mimicry of the Steroid/Thyroid nuclear receptor superfamily. Bioessays 15, 427–432. M, G. J., S, B. R. & J, H. I. (1989). AFP modification of biologic response in estrogen-sensitive tissues: use of in vivo and in vitro models. In: Biological Activities of Alpha-fetoprotein, Vol. II (Mizejewski, G. J. & Jacobson, H. I., eds) pp. 59–74. Boca Raton, FL: CRC Press. M, G. J., V, M. & J, H. I. (1983). Estradiol-activated a-fetoprotein suppresses the uterotrophic response to estrogens. Proc. natn. Acad. Sci. U.S.A. 80, 2733–2737. M, G. J., K, J. F. & S, R. P. (1990). Separation of the estrogen-activated growth regulatory forms of alphafetoprotein in mouse amniotic fluid. Biol. Reprod. 42, 887–898. M, G. J. (1985). New insights into AFP structure and function: potential biomedical applications. In: Alphafetoprotein and Congenital Disorders (Mizejewski, G. J. & Porter, I. H. eds.) pp. 5–34. Orlando, FL: Academic Press. M, G. J. & W, A. S. (1989). Alpha-fetoprotein can regulate growth in the uterus of the immature and adult ovariectomized mouse. J. Reprod. Fertil. 85, 177–185. M, G. J. & B-B, M. M. (1987). Release of AFP from serine protease inhibitors in serum and tissue cytosols. In: Biological Activities of Alpha-fetoprotein, Vol. II (Mizejewski, G. J. & Jacobson, H. I. eds) pp. 162–179. Boca Rato, FL: CRC Press. M, R., T, T., W, T. G., L, B. M. & L, M. P. (1993). Monoclonal antibodies directed against a widespread oncofetal antigen: the alpha-fetoprotein receptor. Tumor Biol. 14, 116–130. M, H., B, G. & P, D. (1993). Signal sequence region of mitochondrial precursor proteins binds to mitochondrial import receptor. Proc. natn. Acad. Sci. U.S.A. 90, 3358–3362. N, J., V, M. J., G, A. F. & U, J. (1985). Cell type specific receptors for AFP in a mouse T-lymphoma cell line. Proc. natn. Acad. Sci. U.S.A. 82, 3301–3304. N, D. D. & F, D. J. (1990). An N-ethylmaleimide-sensitive cytosolic factor necessary for protein import: Requirement in signal-mediated binding to the nuclear pore. J. Cell. Biol. 100, 547–557. N, S., M, H., Y, H., Y, R. & S, M. (1991). Localization of the estrogen-binding site of a-fetoprotein in the chimeric human-rat proteins. Proc. natn. Acad. Sci. U.S.A. 88, 3102–3105. O, E., H, L. M., & M, A. (1993). Kinetics of glucocorticoid receptor phosphorylation in intact cells. J. biol. Chem. 268, 7779–7784. P, D. & Y, K. R. (1987). Two signals mediate hormone-dependent nuclear localization of the glucocorticoid receptor. EMBO J. 6, 3333–3340. P, D., K, V., C, P. & Y, K. R. (1990). Signal transduction by steroid hormones: nuclear localization is differentially regulated in estrogen and glucocorticoid receptors. Cell. Reg. 1, 291–299. P, W. B., C, M. J., S, L. F. & O, J. K. (1993). The HSP 56 immunophilin component of steroid receptor heterocomplexes: Could this be the elusive nuclear localization signal binding protein? J. Steroid Biochem. molec. Biol. 46, 269–279. R, A., B, G. & W, R. W. (1993). NupL155 is a novel nuclear pore complex protein that contains neither repetitive sequence motifs nor reacts with WGA. J. Cell. Biol. 121, 1–9. R, T. A. (1991). Protein transport across the endoplasmic reticulum membrane: facts, models, mysteries. FASEB J. 5, 2792–2798. R, M., B, W. & G, U. (1991). Protein components of the non-activated glucocorticoid receptor. J. biol. Chem. 266, 24601–24605. R, B. (1989). Nuclear location signal-mediated protein transport. Biochem. Biophys. Acta. 1008, 263–280.
R, E. D., O’D, A. L. & K, R. J. (1991). Protein–protein interactions involving erbA superfamily receptors: through the TRAP door. Molec. Cell. Endocrinol. 78, C83–C88. R, J. E. (1989). Polypeptide chain binding proteins: catalysts of protein folding and related processes in cells. Cell 59, 591–601. R, A. H., S, V., F, M., B, G. & V, J. (1981). Relation of glucose to alphafetoprotein in amniotic fluid. Obs. & Gyn. 57, 310–312. S, E. J., Z, M. & D, D. S. (1983). M, G. J. & J, H. I. Detection and measurement of alpha-fetoprotein in human breast cancer cytosol after treatment with 0.4 M potassium chloride. Cancer Res. 43, 3739–3741. S, E. J. & S, J. R. (1976). Intracellular synthesis of alpha-fetoprotein and fibrinogen without secretion by zajdela rat hepatoma cells. Cancer Res. 36, 3203–3205. S, G. (1993). The protein import machinery of mitochondria. Protein Sci. 2, 141–146. S, G. & G, M. C. (1992). Activation of Kb-specific proteins by estradiol. Proc. natn. Acad. Sci. U.S.A. 89, 10628–10632. S, P. A. (1991). How proteins enter the nucleus. Cell 64, 489–497. S, J. A., S, M., L, M. V., F, F. S. & W, E. M. (1991). Transcriptional activation and nuclear targeting signals of the human androgen receptor. J. biol. Chem. 266, 510–518. S, D. E. & T, D. O. (1993). Steroid receptors and their associated proteins. Molec. Endocrinol. 7, 4–11. S, B. & W, H. J. (1993). The amino-terminal domain of the lamin-B receptor is a nuclear envelope targeting signal. J. Cell. Biol. 120, 1093–1100. S, U. K., R, C. J. & D, M. R. (1987). Heat-induced expression of albumin during early states of rat embryo development. Molec. Cell. Biol. 7, 4599–4602. S, U. K. & R, C. J. (1994). Altered expression of alpha-fetoprotein and albumin in early embryonic rat liver upon heat shock. Biochem. Molec. Biol. Int. 33, 21–28. S, C. M., D’O, P, M. K. & H, J. A. (1990). Primary sequence and heterologous expression of nuclear pore glycoprotein. p. 62. J. Cell. Biol. 110, 1861–1871. S, U. & S, P. (1992). Nucleocytoplasmic traffic of proteins. Eur. J. Cell. Biol. 59, 1–11. S, J. & B, G. (1993). A nuclear pore complex protein that contains zinc finger motifs, binds DNA and faces the nucleoplasm. Cell, 72, 29–38. S, Y., Z, C. Q. Y. & A, E. (1992). Isolation and characterization of a specific alpha-fetoprotein receptor on human monocytes. J. Clin. Invest. 90, 1530–1536. T, Y. S. (1965). Content of embryo-specific alpha-globulin in the blood serum of the human foetus; newborn, and adult man in primary cancer of the liver. Vop. Khim. SSSR 11, 20–24. T, T. B. (1977). Structure and Function of alpha-fetoprotein. A. Rev. Med. 28, 453–465. T, T. C. (1993). New Model for 70 kDa heat-shock proteins’ potential mechanism of function. FEBS Lett. 323, 1–3. T, P., P, A., B, M., J, T., V, P. & Y, T. (1993). Nuclear progesterone receptor is mainly heat shock protein 90-free in vivo. Proc. natn. Acad. Sci. U.S.A. 90, 5848–5852. U, J., N, J. & L, J. (1986). Alpha-fetoprotein mediated transfer of arachidonic acid into cultural cloned cells derived from a rat rhabdomyosarcoma. J. biol. Chem. 262, 3579–3585. U, J., F-C, C., V, M. J., P, A. & G, M. (1984). Incorporation of alpha-fetoprotein by the MCF-7 human breast cancer cell line. Tumor Biol. 5, 41–51. W, W. & M, E. (1991). Superfamily of steroid nuclear receptors: positive and negative regulators of gene expression. FASEB J. 5, 2243–2249. W, S. R., R, M. P. & B, G. (1992). A new family of yeast nuclear pore complex proteins. J. Cell. Biol. 119, 705–723.
/ W, S. T. & G, S. (1993). Signal transduction and nuclear targeting: regulation of transcription factor activity by subcellular localization. J. Cell. Sci. 104, 949–955. W, F. G., N, M., & S, C. (1992). Candidate protonocogene bcl-3 encodes a subunit-specific inhibitor of transcription factor NF-kB. Nature, Lond. 358, 597–599. Y, L., K, P. & L, R. E. (1989). Identification of four nuclear transport signal-binding proteins that interact with diverse transport signals. Molec. Cell. Biol. 9, 3028–3036.
113
Y, F. & O, M. (1992). The gene for the rat heat-shock cognate, hsc 70, can suppress oncogene-mediated transformation. Cell Growth Diff. 3, 803–809. Y, T., B, M. T., B, M., G, H. & C, P. (1992). Cooperation of proto-signals for nuclear accumulation of estrogen and progesterone receptors. EMBO J. 11, 3681–3694. Z, K. V., A, F. & F, E. (1991). A hydrophobic protein sequence can override a nuclear localization signal independently of protein context. Molec. Cell. Biol. 11, 5137–5146.