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Receptor signalling and the regulation of endocytic membrane transport
549
Receptor signalling and the regulation of endocytic membrane transport Matthew NJ Seaman, Christopher G Burd and Scott D Emr* Vesicle-mediated membrane traffic has long been considered to be a constitutive process that is not burdened by layers of regulation. This contrasts with transmembrane signalling systems at the plasma membrane which relay information (i.e. extracellular stimuli) from the cell surface to the cytoplasm via a myriad of different protein-protein interactions and second messenger cascades. An accumulation of recent evidence, however, now suggests that signal-transduction pathways also play a critical role in the regulation of protein and membrane trafficking. In particular, the analysis of the signalling pathways initiated by receptor tyrosine kinases at the plasma membrane has yielded new insights into the molecular mechanisms of endocytosis. In addition, recent evidence has suggested potential new roles for two previously characterized vesicle coat proteins in a membrane traffic route that is regulated via cell surface receptor signalling.
Address Division of Cellular and Molecular Medicine and Howard Hughes Medical Institute, University of California at San Diego, School of Medicine, La Jolla, CA 920?5-0668, USA ('Author for correspondence) Current Opinion in Cell Biology 1996, 8:549-556
© Current Biology Ltd ISSN 0955-0674 Abbreviations AP adaptor complex EGF epidermal growth factor EGFR EGF receptor EH epsl 5 homology FcTR Fcy IgG receptor IRS-1 insulin receptor substrate-1 MAPK mitogen-activated protein kinase MARCKS myristoylated,alanine-rich, C-kinase substrate PDGF ptatelet-derived growth factor PDGFR PDGF receptor PI phosphoinositide Pl 3-P PI 3-phosphate P! 3,4-P2 PI 3,4-bisphosphate Pl 3,4,5-P3 PI 3,4,5-trisphosphate Ptdlns phosphatidylinositol SH2 Src homology 2 TGN trans-Golgi network
Introduction Signal transduction is often thought of as a cascade of interactions initiated at the plasma membrane but exerting effects in the nucleus; for instance, the p21Rasactivation pathway leads to stimulation of the mitogenactivated protein kinases (MAPKs) which ultimately activate transcription factors such as Fos and Jun. However,
certain signal-transduction pathways exert their effects in the cytoplasm and have a profound influence upon membrane traffic; for instance, insulin binding stimulates trafficking within the endocytic system. In this review, we will use the term 'signal transduction' to describe a process by which ligand binding to a receptor initiates a series of responses on the opposite side of the membrane. In particular, we examine the role of signalling in the initial steps required for membrane transport, that is, in vesicle formation and cargo-loading within the post-TGN (post-trans-Golgi network) and endocytic pathways. Events downstream of vesicle formation, that is the docking and fusion of vesicles with membranes, and the regulatory mechanisms involved in these steps are comprehensively reviewed elsewhere [1,2]. In addition, the role of signal-transduction pathways in membrane trafficking in polarized epithelial cells has also recently been reviewed [3] and will not be covered here.
Endocytosis Regulation of receptor tyrosine kinase trafficking Binding of epidermal growth factor (EGF) to its tyrosine kinase containing receptor on the plasma membrane results in a striking redistribution of receptor-ligand complexes. Activated receptors first dimerize, concentrate in clathrin-coated pits, and are then internalized via clathrin-coated vesicles for eventual delivery to the lysosome where they are degraded. As a result of this process, termed receptor downregulation, growth factor binding sites are depleted from the plasma membrane and mitogenic effects are attenuated. Downregulation is an important negative regulatory component of receptor tyrosine kinase signalling pathways and defects in this regulation can result in prolonged mitogenic signalling, as is seen in many forms of cancer. Recent progress in understanding the mechanism of receptor downregulation has revealed that endocytosis of the E G F receptors (EGFRs) and of other receptor tyrosine kinases is regulated by the receptors themselves. E G F R has a large carboxy-terminal cytoplasmic domain which contains a tyrosine kinase domain near the transmembrane segment, followed by a region closer to the carboxyl terminus that contains multiple endocytic codes ([4]; see Fig. la). Prior to hormone binding, EGFRs are stable and cycle between the plasma membrane and endosomes at a relatively slow basal rate. Studies employing mutant EGFRs indicate that intrinsic tyrosine kinase activity is required for internalization; a point mutation that abolishes kinase activity results in endocytosis of occupied EGFRs at a slow rate with or without ligand [5"',6"]. Although
550
Membranes and sorting
EGF-induced receptor autophosphorylation is required for stimulated endocytosis of wild-type receptors, ligand dependence and the phosphotyrosine requirement can be bypassed, as mutant receptors lacking the entire kinase domain (but retaining other portions of the cytoplasmic domain) were found to be internalized, with or without EGF, more rapidly than wild-type unoccupied receptors [6’]. The trafficking behavior of mutant EGFRs lacking only the kinase domain differs in several respects from that of wild-type receptors, so it is difficult to determine if the
mutant receptors follow the same endocytic wild-type receptors.
pathway as the
What is the specific function of receptor kinase activity in endocytosis? Recent work suggests that it is required to recruit receptors into clathrin-coated pits, one of the first steps in endocytosis [5”,7]. Several groups have found that the cytoplasmic domain of EGFR, in particular the phosphotyrosine-containing regulatory region, binds the clathrin-associated adaptor complex AP2
Figure 1
Diagrams of
the EGF and PDGF
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(a)
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PDGFR
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trafficking
and/or
with its cytoplasmic
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\ Kinase
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and autophosphorylation.
insert
Carboxyl terminus
domain
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I
EGF leads to receptor dimerization, activation of receptor tyrosine kinase activity
kinase domains
Amino
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transduction are also indicated with circled ‘P’s (b) Unactivated EGFR resides in the plasma membrane as a monomer
Tvrosine
I
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I I
The
activated receptor also phosphorylates several cytosolic proteins, including epsl5. Receptor autophosphorylation may cause a conformational change
in
the cytoplasmic domain of the receptor which leads to stoichiometric binding of AP2 that may be modulated by eps15. These interactions lead to receptor endocytosis via clathrin-coated receptor downregulation.
pits and
interaction
Clathrin-coated
AP2 + Downregulation
pit
Receptor signalling and the regulation of endocytic membrane transport Seaman, Burd and Emr 551
[8-12]. Whether or not full AP2-binding activity requires tyrosine phosphorylation remains a controversial issue, although it is clear that receptor phosphorylation is an important factor. Matters are further complicated by the observation that activated E G F R phosphorylates not only itself but also a small group of proteins including epslS, which is phosphorylated rapidly by activated EGFR and platelet-derived growth factor receptor (PDGFR) [13]. Epsl5 contains a newly described conserved protein-binding domain, termed the epsl5 homology (EH) domain, which is found in a small number of proteins including End3p, a yeast protein required for endocytosis of ~ factor pheromone [14°]. Interestingly, a small fraction of epsl5 has been found in a complex with AP2 [15**], suggesting that it could recruit, stabilize or activate this pool of AP2. Previous studies have hinted at the existence of a tyrosine-phosphorylated cytosolic factor that is possibly required for E G F R internalization [16], and it is tempting to speculate that epsl5 may fulfill this requirement. However, phosphorylation of epsl5 by EGFR is not required for epslS-AP2 complex formation [15°*], so the significance of this association for stimulated endocytosis of the E G F R is not apparent. As both epsl5 and End3p are involved in endocytosis, other EH domain containing proteins may function in endocytosis or at other sites of membrane trafficking. There are at least two possible models that could account for stimulated E G F R endocytosis following ligand binding. Receptor autophosphorylation could cause a conformational change in the cytoplasmic domain of the receptor [17], exposing a cryptic endocytic code for binding a factor(s) required for stimulated endocytosis. Alternatively, or additionally, activated receptors might phosphorylate a different protein, such as epsl5, that could modulate AP2 function. Binding of AP2 would be expected to recruit clathrin to receptor patches or could lead to recruitment of activated receptors to coated pits, resulting in receptor endocytosis (see Fig. lb). Thus far, however, clathrin has not been detected in EGFR immunoprecipitates. Phosphorylation is not the only covalent modification involved in regulated endocytosis of plasma membrane receptors. T h e yeast G protein coupled receptor for factor pheromone, Ste2p, was recently shown to require addition of a single ubiquitin molecule for its ligand-stimulated endocytosis and degradation in the vacuole/lysosome [18°°]. In mutant yeast strains that are unable to ubiquitinate Ste2p, the receptor was not internalized from the plasma membrane and was stabilized [18°*]. Ubiquitination may prove to be a modification generally required for vacuolar targeting of plasma membrane proteins, as other plasma membrane proteins require ubiquitination for their normal trafficking and stability [19,20]. A signalling role for ubiquitin may not be limited to yeast. In mammalian cells, the tyrosine kinase activity of E G F R and c-Kit (a member of the P D G F R family)
is required for their downregulation and, surprisingly, for their ligand-stimulated polyubiquitination [21,22]. It is not yet known if these receptors are degraded in the lysosome or by the proteasome. Together, these findings suggest a previously unappreciated role for ubiquitin in regulated endocytosis and vacuolar/lysosomal targeting, apart from its well characterized role in proteasome-mediated protein degradation. It is not yet clear whether this modification is directly recognized by the endocytic machinery or whether it causes a conformational change that exposes a sorting signal. Phosphatidylinositol 3-kinases and protein sorting
Recent findings have suggested important regulatory roles for phosphorylated products of phosphatidylinositol in membrane trafficking. In yeast cells, a phosphatidylinositol (Ptdlns) specific 3-kinase encoded by the VPS34 gene has been shown to be required for the sorting of vacuolar/lysosomal hydrolases [23*]. Membrane association of Vps34p and activation of its Ptdlns 3-kinase activity are regulated by a Ser/Thr protein kinase, Vpsl5p, and a model has been proposed whereby this complex regulates vesicle budding and/or cargo loading in the late Golgi [23°,24,25]. In permeabilized rat PC12 cells, a Ptdlns 4-phosphate 5-kinase is required for the priming and exocytosis of secretory granules [26*]. Mammalian cells contain numerous phosphoinositide (PI) 3-kinase isoforms, including a Vps34p homolog [27]; however, the functions of these enzymes are not known. It is clear that different classes of PI 3-kinases, distinguished by their ability to utilize different substrates, can lea6 to distinct cellular responses, suggesting that different signalling pathways may be mediated by different PI products (reviewed in [28",29]). PI 3-kinase activity has been implicated in trafficking events subsequent to receptor internalization, possibly in transport from endosomes to the lysosome. In one set of experiments, tyrosines in the cytoplasmic tail of PDGFR that are phosphorylated in response to P D G F binding were systematically mutated, and the effect of these mutations on P D G F R trafficking was assessed [30]. These mutations interfere with the binding of a variety of Src homology 2 (SH2) domain containing signalling molecules, including the regulatory subunit of PI 3-kinase (p85), the GTPase-activating protein (GAP) of p21Ras, phospholipase C-~' and the phosphotyrosine phosphatase Syp, which all bind activated PDGFR. Specific changes in the tyrosine residues known to bind PI 3-kinase blocked delivery of P D G F R to lysosomes and stabilized the receptor, whereas other mutant receptors trafficked normally [30]. Inhibition of PI 3-kinase activity by the drug wortmannin also blocked lysosomal trafficking of PDGFR and resulted in recycling of the receptor to the plasma membrane, suggesting that PI 3-kinase is required to sort activated P D G F R to the lysosomal delivery pathway [31,32]. Experiments addressing a potential role(s) for PI 3-kinase activity in trafficking of the E G F R have not been reported; however,
552 Membranes and sorting
like PDGFR, the kinase activity of E G F R is required both to sort occupied E G F R to internal vesicles of multivesicular bodies and for subsequent receptor degradation in lysosomes [33,34]. Wortmannin has also been shown to cause mis-sorting of cathepsin D, a lysosomal protease that is sorted from the secretory pathway in the T G N by the mannose 6-phosphate receptor [35,36]. These results imply a role for mammalian PI 3-kinase(s) in endosomal and/or T G N function. T h e observation that PI 3-kinase can modulate Rab5-mediated fusion of endosomes in vitro is consistent with these results [37]. Clearly, localization of the different PI kinase isoforms remains an issue of central importance for understanding the respective roles of the isoforms in mitogenic signalling and regulation of membrane traffic.
Signal-mediated GLUT-4 trafficking One of the most extensively studied signal-transduction pathways is the signalling cascade that results from the binding of insulin to its receptor. Insulin has a dual role: it is both a weak mitogen and a regulator of glucose metabolism. In adipose and muscle tissue, insulin causes a rapid and acute stimulation of glucose uptake. This is mediated primarily by the G L U T - 4 glucose transporter which is sequestered intracellularly within adipocytes and muscle cells and is localized to a portion of the endocytic system. In muscle and fat cells, G L U T - 4
cycles slowly between this endocytic compartment and the plasma membrane, but, upon binding of insulin to the insulin receptor, there is a increase in trafficking from an endocytic 'storage' compartment to the plasma membrane, causing G L U T - 4 to be rapidly translocated to the plasma membrane where it can exert its effect upon glucose uptake (reviewed in [38]; see Fig. 2). T h e signalling pathway that is activated after insulin binds to its receptor is very complex and involves a host of signalling molecules, including the insulin receptor substrate-1 (IRS-1), Grb2-SOS, p21Ras and the p85-p110 form of PI 3-kinase (see [39,40] for reviews). Evidence is emerging to suggest that essentially two divergent signalling pathways result from activation of the insulin receptor and that these pathways separately regulate the mitogenic response and the GLUT-4-trafficking pathway, respectively [41°°,42-44]. After binding insulin, the insulin receptor is rapidly autophosphorylated at multiple tyrosine residues in its cytoplasmic tail domain; these residues provide the binding sites for several SH2 domain containing proteins [45,46"]. Protein-protein interactions mediated by specific phosphotyrosines and SH2 domains form the basis of many of the interactions in this signalling pathway (reviewed in [40]). Although the interactions that are required for
Figure 2 A schematic diagram of some of the membrane trafficking events that occur
either after insulin binding or after activation of Fcy receptors by binding of IgG-coated antigen. Insulin binding to the insulin receptor somehow results in the increased translocation of GLUT-4, via vesicles which bud from an endosomal compartment, to the plasma membrane and in the internalization of the insulin receptor into clathrin-coated pits for subsequent delivery to endosomes and then lysosomes. Phagocytosis of IgG-coated antigen occurs after Fcy-receptor activation which in turn results in a signalling cascade that ultimately causes a rearrangement of the actin cytoskeleton at the site of antigen internalization. Phagocytosed antigen is eventually degraded in structures called phagolysosomes. Common to both these events is receptor phosphorylation. PI 3-kinase has been implicated in many events with the post-TGN and endocytic trafficking pathways, and may function at various points to direct specific proteins into vesicles or facilitate vesicle formation through activation of vesicle coat proteins.
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Receptor signalling and the regulation of endocytic membrane transport Seaman,Burd and Emr 553
the mitogenic effects of insulin are starting to be characterized, components of the machinery required for the translocation of G L U T - 4 to the plasma membrane remain to be formally identified. A strong candidate protein for acting directly in the GLUT-4-translocation pathway is p85-p110 PI 3-kinase. Microinjection of a fusion protein containing the SH2 domain of p85 was able to block both the insulin-stimulated translocation of G L U T - 4 and, consequently, the increase in glucose uptake [41"]. T h e PI 3-kinase inhibitor wortmannin potently blocks the translocation of G L U T - 4 to the plasma membrane [47-49], and dominant-negative mutants of the p85 subunit of PI 3-kinase which fail to bind p l l 0 (the catalytic domain of PI 3-kinase) also block insulin-stimulated glucose uptake but do not block activation of p21Ras [42,49]. Presently, however, the evidence indicates that p85-p110 PI 3-kinase is also involved in the mitogenic pathway [50] and is not exclusively part of the GLUT-4-translocation machinery. This may reflect the fact that PI 3-kinase can catalyze the production of more than one phosphoinositide product (reviewed in [28"]). A simple model therefore is that perhaps PI 3-phosphate (PI 3-P) and/or PI 3,4-bisphosphate (PI 3,4-P 2) is involved in G L U T - 4 translocation, and that PI 3,4,5-trisphosphate (PI 3,4,5-P 3) is required in the mitogenic pathway. Consistent with this model is the finding that PI 3,4,5-P3 can modulate the interaction between phosphotyrosines and SH2 domains ([51"]; there are many more SH2 domain containing proteins believed to be involved in the mitogenic signalling pathway than there are in the GLUT-4-translocation pathway). T h e question remains, however, as to what machinery is involved in the rapid translocation of G L U T - 4 to the plasma membrane. It is now widely accepted that cytoplasmic protein complexes called 'coats' mediate vesicle formation from the compartments that constitute the secretory and endocytic pathways (reviewed in [52,53]). T h e recruitment of coats from the cytoplasm to the membrane involves small GTPases of the ADP-ribosylation factor (ARF) and SAR families. Thus far, there are four distinct coats which have been isolated and characterized, namely clathrin together with either AP1 or AP2, coatomer/COPI, and COPII. Until recently; however, there was no candidate coat protein to mediate the translocation of G L U T - 4 from an endocvtic compartment to the plasma membrane. Recent reports have now provided two possible candidates. One is coatomer/COPI which has recently been implicated in trafficking within the endocytic systcm [54°,55] and which also has a recently established role in the recycling of proteins from the Golgi apparatus to the endoplasmic reticulum [56]. T h e other candidate is clathrin together with an adaptor complex which presumably has yet to be identified. Using immunoelectron microscopy, clathrin was recently localized to a compartment accessible to endocytosed molecules [57°°]. Clathrin is known to act at both the plasma membrane and the T G N to mediate the formation of endocytic vesicles and vesicles required for the delivery
of newly synthesized lysosomal enzymes, respectively; thus, it seems plausible that clathrin may also mediate the formation of GLUT-4-containing vesicles from some part of the endocytic system. A simple hypothesis, therefore, is that upon stimulation by insulin, the budding of vesicles containing G L U T - 4 is upregulated, possibly by stimulation of coat protein recruitment to the endosomal membrane. This ahme may not be sufficient to efficiently deliver G L U T - 4 to the plasma membrane; hence, some form of covalent modification may be required to promotc the loading of G L U T - 4 into vesicles. Presently, however, the mechanism by which the stimulation of coat recruitment could occur has yet to be determined. One possibility is that one of the molecules in the G L U T - 4 ttanslocation signalling pathway acts as a guanine nucleotide exchange factor for an ARF-likc small GTPase. Interestingl3; it has been recently demonstrated that PI 3-kinase can promote GDP for G T P exchange on Rac, another small GTPasc of the p21Ras superfamily [58",59]; this suggests that perhaps PI 3-kinase could act to promote GDP for G T P exchange on an ARF/SAR-Iike small GTPase. This invites speculation as to the role of PI 3-kinases in vesicle coat protein recruitment for GLUT-4-containing vesicles and for other vesicles in the secretory pathway. Although some of the key players in G L U T - 4 translocation are starting to be identified, the process remains something of a 'black box'. Questions regarding the regulation of coat protein assembly/targeting and vesicle formation form a theme throughout the study of membrane traffic, but are particularly pertinent in the case of G L U T - 4 translocation.
Phagocytosis Phagocytosis constitutes another form of ligand uptake from the plasma membrane and is the process by which macrophages and other hematopoietic cells ingest relatively large particles such as bacteria. It differs from endocytosis in that phagocytosis does not employ relatively small vesicles to mediate the internalization process, but instead occurs by large-scale membrane movement which is driven by rearrangement of the actin cytoskeleton close to the plasma membrane. T h e Fcy IgG receptor (FcyR) plays a pivotal role in this process by binding antigens (e.g. bacteria) coated with IgG and initiating a signalling pathway that ultimately leads to rearrangement of the actin cytoskeleton close to the plasma membrane (see Fig. 2). T h e FcyR exists as multiple isoforms; one of the best characterized Fcy receptors is FcyRIIA which is expressed in many hematopoietic cells, including monocvtes neutrophils and B cells (reviewed in [60,61°]). Unlike the insulin receptor and the E G F receptor, the FcyRIIA does not possess intrinsic tyrosine kinase activity that initiates a signalling cascade. Binding of IgG to FcyRIIA results in the phosphorylation of the cytoplasmic domain of the receptor at multiple tyrosine residues which may act as binding sites for SH2 domain
554
Membranes and sorting
containing proteins. Two candidate kinases, p72syk and Fgr, are implicated in the phosphorylation of FcyRIIA [62-64]. Another of the potential targets of these kinases is paxillin, which is phosphorylated after IgG binding to FcyRIIA [63]. Paxillin is a cytoskeleton-associated protein and has therefore been suggested to function in the rearrangement of the actin cytoskeleton that is required for phagocytosis. Also implicated in this role is the protein MARCKS (myristoylated, alanine-rich, C-kinase substrate), which is a substrate for protein kinase C and acts as an actin cross-linking protein [65]. MARCKS associates with nascent phagosomes at the same time as does F-actin. Phosphorylation may regulate the association of MARCKS with phagosomes. As MARCKS is myristoylated at its amino terminus, a modification which allows a protein to associate stably with a membrane, it has been proposed that MARCKS acts in linking actin to the plasma membrane [66"]. Phagocytosis is another cellular process that involves PI 3-kinase activity [67]. p85 is phosphorylated upon FcyR activation, and treatment with wortmannin prevents FcyR-mediated phagocytosis. Presently, however, the role of PI 3-kinase in phagocytosis is not clear.
domain, will play a significant role in the regulatory mechanisms governing membrane traffic. Many important questions clearly remain to be answered and we can anticipate many exciting new developments that will further cement the link between signal transduction and membrane trafficking.
Acknowledgements We thank Sand's" Schmid for critically reading the manuscript. CG Burd is a postdoctoral Fellow of the American Cancer Society. MNJ Seaman is supported by a fellowship from the European Molecular Biology Organisation. SD Emr is an Investigator of the Howard Hughes Medical Institute.
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Conclusions and perspectives Signal transduction and membrane trafficking have, in the past, been regarded as separate disciplines within cell biology, but clearly this is no longer the case. Coat proteins that mediate vesicle formation create the vehicles to transport molecules from one compartment to another, and clearly also play a role in determining the specific cargo that will be conveyed by the vesicles. However, in the case of signalling receptors, for example the EGFR, other mechanisms are present to ensure that the receptor is only targeted to vesicles after ligand binding. One of these mechanisms is phosphorylation of the receptor, which may in fact serve two roles, firstly to initiate a signal-transduction cascade and secondly to target the receptor for internalization. T h e signalling cascade may in itself have two functions, namely to stimulate the pathway which gives rise to the mitogenic effects of the growth factor and to upregulate or modify a specific transport pathway. This appears to be the case for insulin-stimulated G L U T - 4 translocation. Signalling molecules such as PI 3-kinase are now clearly implicated in the selective packaging of cargo into transport vesicles and in the entire trafficking pathway, presumably by recruitment to the membrane/activation of the machinery involved in that pathway. T h e phosphoinositide products of these enzymes may regulate the activity of small GTPases which function in vesicle budding and fusion. T h e proteins involved in this process are only now starting to be identified, and this is the first step in understanding how signal transduction influences membrane traffic. Protein-protein interactions mediated by phosphotyrosines and SH2 domains are clearly important and it now seems likely that other protein-protein interaction domains, for example the EH
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Keller SR, Lienhard GE: Insulin signalling: the role of insulin receptor substrata 1. Trends Cell Bio/1994, 4:115-119.
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White MF, Kahn CR: The insulin signalling system. J Bio/Chem 1994, 269:1-4.
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Benmerah A, Gagnon J, Begue B, Megarbane B, Dautry-Varsat A, Cerf-Bensussan N: The tyrosine kinase substrate eps15 is constitutively associated with the plasma membrane adaptor AP-2. J Ceil Bio/1995, 131:1831-1838. The authors use co-immunoprecipitation and in vitro binding studies to demonstrate that eps15, a protein phosphorylated by EGFR and PDGFR, is associated with the plasma membrane clathrin adaptor complex AP2. 16.
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LamazeC, Baba T, Redelmeier TE, Schmid SL: Recruitment of epidermal growth factor and transferrin receptors into coated pits in vitro: differing biochemical requirements. Mo/Bio/Cell 1993, 4:715-727 Cadena DL, Chan CL, Gill GN: The intracellular tyrosine kinase domain of the epidermal growth factor receptor undergoes a conformational change upon autophosphorylation. J Bio/Chem 1994, 269:260-265.
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Stack JH, DeWald DB, Takegawa K, Emr SD: Vesicle mediated protein transport: regulatory interactions between the Vps15 protein kinase and the Vps34 Ptdlns 3 kinase essential for protein sorting to the vacuole in yeast. J Cell Bio/1995, 129:321-334. The authors demonstrate that the Ptdlns 3-kinase activity of the yeast protein Vps34p is required for the sorting of vacuolar hydrolases. Furthermore, it is shown that the Ptdlns 3-kinase activity of Vps34p is regulated by the protein kinase encoded by VPS15 which is also required for vacuolar protein sorting. A model is presented to explain the sorting of vacuolar hydrolases in the late Golgi. 24.
Stack JH, Horazdovsky B, Emr SD: Receptor-mediated protein sorting to the vacuole in yeast. Annu Rev Cell Bio11995, 11:1-33.
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Horazdovsky BF, DeWald DB, Emr SD: Protein transport to the yeast vacuole. Curr Opin Cell Bio11995, 7:544-551.
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Hay JC, Fisette PL, Jenkins GH, Fukami K, Takenawa T, Anderson RA, Martin TH: ATP-dependent inositide dephosphorylation required for Ca2+-activated secretion. Nature 1995, 374:173-177. Using a permeabilized cell based assay, this group had previously identified three activities in rat brain extracts required for regulated secretion of secretory granules, one of which was phosphatidylinositol transfer protein (PtdlnsTP). The purification of a second factor is presented in this paper and shown to be a phosphatidylinositol 4-phosphate 5-kinase (PtdlnsP5K).
41. •.
Haruta T, Morris AJ, Rose DW, Nelson JG, Mueckler M, Olefsky JM: Insulin stimulated GLUT4 translocation is mediated by a divergent intracellular signaling pathway. J Biol Chern 1995, 270:27991-2?994. Using microinjection into single adipocytes, the authors show that inhibition of PI 3-kinase prevents insulin-stimulated GLUT-4 translocation, but that microinjection of oncogenic p21Ras or of p21Ras inhibitors does not. This indicated that GLUT-4 translocation and the mitogenic effects of insulin may be mediated through divergent pathways. 42.
Hara K, Yonezawa Y, Sakaue H, Ando A, Kotani K, Kitamura T, Kitamura Y, Ueda H, Stephens L, Jackson TR et aL: Iphosphatidylinositol 3-kinase activity is required for insulinstimulated glucose transport but not for RAS activation in CHO cells. Proc Nat/Acad Sci USA 1994, 91:7415-7419.
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Van Den Berghe N, Ouwens DM, Maasen JA, Van Maekelenbergh MGH, Sips HCM, Krans HMJ: Activation of the Ras/Mitogen-activated protein kinase signalling pathway alone is not sufficient to induce glucose uptake in 3T3-L1 adipocytes. Mol Cell Biol 1994, 14:2372-2377.
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ReuschJE-B, Bhuripanyo P, Carel K, Leitner JW, Hsieh P, DePaolo D, Draznin B: Differential requirement for p21ras activation in the metabolic signalling by insulin. J Biol Chem 1995, 270:2036-2040.
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Staubs PA, Reichart D, Saltiel AR, Milarski KL, Maegawa H, Berhanu P, Olefsky JM, Seely BL: Localization of the insulin receptor binding sites for the SH2 domain proteins p85, syp and GAP. J Bio/Chem 1994, 269:27186-27192.
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Letourneur F, Gaynor EC, Hennecke S, Demolliere C, Duden R, Emr SD, Riezman H, Cosson P: Coatomer is essential for retrieval of dilysine tagged proteins to the endoplasmic reticulum. Cell 1994, 79:1199-1207.
Ward CW, Gough KH, Rashke M, San Wan S, Tribbick G, Wang J-X: Systematic mapping of potential binding sites for Shc and Grb2 SH2 domains on the insulin receptor substrate1 and the receptors for insulin, epidermal growth factor, platelet-derived growth factor and fibroblast growth factor. J Biol Chem 1996, 271:5603-5609. An extensive study of the interactions of both Shc and Grb2 with various growth factor receptors and IRS-1 using phosphotyrosine peptides.
Stoorvogel W, Oorschot V, Geuze HJ: A novel class of clethrincoated vesicles budding from endosomes. J Cell Bio11996, 132:21-33. Describes the localization of clathrin on endosomes by immunogeld electron microscopy. Clathrin buds appeared to be restricted to clathrin-coated regions, and use of the drug brefeldin A indicated that the clathrin present was involved in transport from endesomes, possibly for the recycling of transferrin receptor.
4?.
Okada T, Kawano Y, Sakakibara T, Hazeki O, Ui M: Essential role of the phosphatidylinositol 3-kinase in insulin-induced glucose transport and antilipolysis in rat adipocytes. J Bio/Chem 1994, 269:3568-3573.
58. •
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Clarke JF, Young PW, Yonezawa K, Kasuga M, Holman GD: Inhibition of the translocation of GLUT-1 and GLUT-4 in 3T3L1 cells by the phosphatidyl 3-kinase inhibitor, wortmannin. Biochem J 1994, 300:631-635.
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KotaniK, Carozzi AJ, Sakaue H, Hara K, Robinson I.J, Clark SF, Yonezawa K, James DE, Kasuga M: Requirement for phosphoinositide 3-kinase in insulin-stimulated GLUT4 translocation in 3T3-L1 adipocytes. Biochem Biophys Res Commun 1995, 209:343-348.
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Cheatham B, Vlahos CJ, Cheatham L, Wang BJ, Kahn CR: Phosphatidylinositol 3-kinase activation is required for insulin stimulation of pp70 $6 kinase, DNA synthesis and glucose transporter translocation. Me/Cell B/el 1994, 14:4902-4911.
RamehLE, Chen C-S, Cantley LC: Phosphatidylinositol (3,4,5)P 3 interacts with SH2 domains and modulates PI 3-kinase association with tyrosine phosphorylated proteins. Ceil 1995, 83:821-830. Provides evidence that PI 3-kinase can modulate its own interaction with phosphotyrosine-containing proteins via its product PI 3,4,5-P 3. pp60 c'src was also shown to bind to PI 3,4,5-P 3, implying that PI 3,4,5-P3 may also act as a site of recruitment of SH2 domain containing proteins.
5?. •-
Hawkins PT, Eguinoa A, eui R-G, Stokoe D, Cooke F-I', Waiters R, Wennstrom S, Claesson-Welsh L, Evans T, Symons M, Stephens L: PDGF stimulates an increase in GTP-Rac via activation of phosphoinositide 3-kinase. Curt Bio/1995, 5:393-403. Stimulation of PI 3-kinase by binding of PDGF to the PDGFR results in an increase in the ratio of Rac.GTP to Rac-GDP. This increase in dependent upon PI 3,4,5-P 3. 59.
ParkerPJ: PI 3-kinase puts GTP on the rac. Curt Bio/1995, 5:577-579.
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Lin C-T, Shen Z, Bores P, Unkeless JC: Fc receptor-mediated signal transduction. J C/in Immun 1994, 14:1-13.
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Indik ZK, Park J-G, Hunter S, Schreiber AD: The molecular dissection of Fc'y receptor mediated phagocytosis. Blood 1995, 86:4389-4399. A comprehensive review of the Fc receptor and its role in phagocytosis. 62.
Agarwal A, Salem P, Robbins KC: Involvement of p72syk, a protein-tyrosine kinase, in Fcy receptor signalling. J Bie/Chem 1993, 268:15900-15905.
63.
Greenberg S, Chang P, Silverstein SC: Tyrosine phosphorylation of the y subunit of Fcy receptors, P72syk, and paxillin during Fc receptor-mediated phagocytosis in macrophages. J Bio/ Chem 1994, 269:3897-3902.
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HarnadaF, Aoki M, Akiyama T, Toyoshima K: Association of immunoglobulin G Fc receptor II with a Src-like proteintyrosine kinase Fgr in neutrophils. Proc Nat/Acad Sci USA 1993, 90:6305-6309.
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Hartwig JH, Thelen M, Rosen A, Janmey PA, Nairn AC, Aderem A: MARCKS is an actin filament crosslinking protein regulated by protein kinase C and calcium-calmodulin. Nature 1992, 356:618-622.
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SchekmanR, Orci L: Coat proteins and vesicle budding. Science 1996, 271:1526-1533.
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Schmid SL, Damke H: Coated vesicles: e diversity of form and function. FASEB J 1995, 9:1445-1453.
54. •
Whitney JA, Gomez M, Sheff D, Kreis TE, Mellman I: Cytoplasmic coat proteins involved in endosome function. Ceil 1995, 83:703-? 13. This paper, together with [55], shows that COPI, a coat protein previously shown to be associated with the cis-Golgi membrane, is also associated with endosomes and may function in transport from early to late endosomes. 55.
Aniento F, Gu F, Patton RG, Gruenberg J: An endosomal I~COP is implicated in the pH-dependent formation of transport vesicles destined for late endosomes. J Ceil Bio/1996, 133:29-41.
66. •
Alien bAH, Aderem A: A role for MARCKS, the c~ isozyme of protein kinase C and myosin I in zymosan phagocytosis by macrophages. J Exp Med 1995, 182:829-840. A detailed study of MARCKS localization by immunofluorescence microscopy, showing that MARCKS colocalized with nascent and maturing phagosomes. 6?.
NinomiyaN, Hazeki K, Fukui Y, Seya T, Okada T, Hazeki O, Ui M: Involvement of phosphatidylinositol 3-kinase in Fcy receptor signalling. J Bio/Chem 1994, 269:22732-22?3?.
Receptor signalling and the regulation of endocytic membrane transport Matthew NJ Seaman, Christopher G Burd and Scott D Emr* Vesicle-mediated membrane traffic has long been considered to be a constitutive process that is not burdened by layers of regulation. This contrasts with transmembrane signalling systems at the plasma membrane which relay information (i.e. extracellular stimuli) from the cell surface to the cytoplasm via a myriad of different protein-protein interactions and second messenger cascades. An accumulation of recent evidence, however, now suggests that signal-transduction pathways also play a critical role in the regulation of protein and membrane trafficking. In particular, the analysis of the signalling pathways initiated by receptor tyrosine kinases at the plasma membrane has yielded new insights into the molecular mechanisms of endocytosis. In addition, recent evidence has suggested potential new roles for two previously characterized vesicle coat proteins in a membrane traffic route that is regulated via cell surface receptor signalling.
Address Division of Cellular and Molecular Medicine and Howard Hughes Medical Institute, University of California at San Diego, School of Medicine, La Jolla, CA 920?5-0668, USA ('Author for correspondence) Current Opinion in Cell Biology 1996, 8:549-556
© Current Biology Ltd ISSN 0955-0674 Abbreviations AP adaptor complex EGF epidermal growth factor EGFR EGF receptor EH epsl 5 homology FcTR Fcy IgG receptor IRS-1 insulin receptor substrate-1 MAPK mitogen-activated protein kinase MARCKS myristoylated,alanine-rich, C-kinase substrate PDGF ptatelet-derived growth factor PDGFR PDGF receptor PI phosphoinositide Pl 3-P PI 3-phosphate P! 3,4-P2 PI 3,4-bisphosphate Pl 3,4,5-P3 PI 3,4,5-trisphosphate Ptdlns phosphatidylinositol SH2 Src homology 2 TGN trans-Golgi network
Introduction Signal transduction is often thought of as a cascade of interactions initiated at the plasma membrane but exerting effects in the nucleus; for instance, the p21Rasactivation pathway leads to stimulation of the mitogenactivated protein kinases (MAPKs) which ultimately activate transcription factors such as Fos and Jun. However,
certain signal-transduction pathways exert their effects in the cytoplasm and have a profound influence upon membrane traffic; for instance, insulin binding stimulates trafficking within the endocytic system. In this review, we will use the term 'signal transduction' to describe a process by which ligand binding to a receptor initiates a series of responses on the opposite side of the membrane. In particular, we examine the role of signalling in the initial steps required for membrane transport, that is, in vesicle formation and cargo-loading within the post-TGN (post-trans-Golgi network) and endocytic pathways. Events downstream of vesicle formation, that is the docking and fusion of vesicles with membranes, and the regulatory mechanisms involved in these steps are comprehensively reviewed elsewhere [1,2]. In addition, the role of signal-transduction pathways in membrane trafficking in polarized epithelial cells has also recently been reviewed [3] and will not be covered here.
Endocytosis Regulation of receptor tyrosine kinase trafficking Binding of epidermal growth factor (EGF) to its tyrosine kinase containing receptor on the plasma membrane results in a striking redistribution of receptor-ligand complexes. Activated receptors first dimerize, concentrate in clathrin-coated pits, and are then internalized via clathrin-coated vesicles for eventual delivery to the lysosome where they are degraded. As a result of this process, termed receptor downregulation, growth factor binding sites are depleted from the plasma membrane and mitogenic effects are attenuated. Downregulation is an important negative regulatory component of receptor tyrosine kinase signalling pathways and defects in this regulation can result in prolonged mitogenic signalling, as is seen in many forms of cancer. Recent progress in understanding the mechanism of receptor downregulation has revealed that endocytosis of the E G F receptors (EGFRs) and of other receptor tyrosine kinases is regulated by the receptors themselves. E G F R has a large carboxy-terminal cytoplasmic domain which contains a tyrosine kinase domain near the transmembrane segment, followed by a region closer to the carboxyl terminus that contains multiple endocytic codes ([4]; see Fig. la). Prior to hormone binding, EGFRs are stable and cycle between the plasma membrane and endosomes at a relatively slow basal rate. Studies employing mutant EGFRs indicate that intrinsic tyrosine kinase activity is required for internalization; a point mutation that abolishes kinase activity results in endocytosis of occupied EGFRs at a slow rate with or without ligand [5"',6"]. Although
550
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EGF-induced receptor autophosphorylation is required for stimulated endocytosis of wild-type receptors, ligand dependence and the phosphotyrosine requirement can be bypassed, as mutant receptors lacking the entire kinase domain (but retaining other portions of the cytoplasmic domain) were found to be internalized, with or without EGF, more rapidly than wild-type unoccupied receptors [6’]. The trafficking behavior of mutant EGFRs lacking only the kinase domain differs in several respects from that of wild-type receptors, so it is difficult to determine if the
mutant receptors follow the same endocytic wild-type receptors.
pathway as the
What is the specific function of receptor kinase activity in endocytosis? Recent work suggests that it is required to recruit receptors into clathrin-coated pits, one of the first steps in endocytosis [5”,7]. Several groups have found that the cytoplasmic domain of EGFR, in particular the phosphotyrosine-containing regulatory region, binds the clathrin-associated adaptor complex AP2
Figure 1
Diagrams of
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kinase domains
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I I
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the cytoplasmic domain of the receptor which leads to stoichiometric binding of AP2 that may be modulated by eps15. These interactions lead to receptor endocytosis via clathrin-coated receptor downregulation.
pits and
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pit
Receptor signalling and the regulation of endocytic membrane transport Seaman, Burd and Emr 551
[8-12]. Whether or not full AP2-binding activity requires tyrosine phosphorylation remains a controversial issue, although it is clear that receptor phosphorylation is an important factor. Matters are further complicated by the observation that activated E G F R phosphorylates not only itself but also a small group of proteins including epslS, which is phosphorylated rapidly by activated EGFR and platelet-derived growth factor receptor (PDGFR) [13]. Epsl5 contains a newly described conserved protein-binding domain, termed the epsl5 homology (EH) domain, which is found in a small number of proteins including End3p, a yeast protein required for endocytosis of ~ factor pheromone [14°]. Interestingly, a small fraction of epsl5 has been found in a complex with AP2 [15**], suggesting that it could recruit, stabilize or activate this pool of AP2. Previous studies have hinted at the existence of a tyrosine-phosphorylated cytosolic factor that is possibly required for E G F R internalization [16], and it is tempting to speculate that epsl5 may fulfill this requirement. However, phosphorylation of epsl5 by EGFR is not required for epslS-AP2 complex formation [15°*], so the significance of this association for stimulated endocytosis of the E G F R is not apparent. As both epsl5 and End3p are involved in endocytosis, other EH domain containing proteins may function in endocytosis or at other sites of membrane trafficking. There are at least two possible models that could account for stimulated E G F R endocytosis following ligand binding. Receptor autophosphorylation could cause a conformational change in the cytoplasmic domain of the receptor [17], exposing a cryptic endocytic code for binding a factor(s) required for stimulated endocytosis. Alternatively, or additionally, activated receptors might phosphorylate a different protein, such as epsl5, that could modulate AP2 function. Binding of AP2 would be expected to recruit clathrin to receptor patches or could lead to recruitment of activated receptors to coated pits, resulting in receptor endocytosis (see Fig. lb). Thus far, however, clathrin has not been detected in EGFR immunoprecipitates. Phosphorylation is not the only covalent modification involved in regulated endocytosis of plasma membrane receptors. T h e yeast G protein coupled receptor for factor pheromone, Ste2p, was recently shown to require addition of a single ubiquitin molecule for its ligand-stimulated endocytosis and degradation in the vacuole/lysosome [18°°]. In mutant yeast strains that are unable to ubiquitinate Ste2p, the receptor was not internalized from the plasma membrane and was stabilized [18°*]. Ubiquitination may prove to be a modification generally required for vacuolar targeting of plasma membrane proteins, as other plasma membrane proteins require ubiquitination for their normal trafficking and stability [19,20]. A signalling role for ubiquitin may not be limited to yeast. In mammalian cells, the tyrosine kinase activity of E G F R and c-Kit (a member of the P D G F R family)
is required for their downregulation and, surprisingly, for their ligand-stimulated polyubiquitination [21,22]. It is not yet known if these receptors are degraded in the lysosome or by the proteasome. Together, these findings suggest a previously unappreciated role for ubiquitin in regulated endocytosis and vacuolar/lysosomal targeting, apart from its well characterized role in proteasome-mediated protein degradation. It is not yet clear whether this modification is directly recognized by the endocytic machinery or whether it causes a conformational change that exposes a sorting signal. Phosphatidylinositol 3-kinases and protein sorting
Recent findings have suggested important regulatory roles for phosphorylated products of phosphatidylinositol in membrane trafficking. In yeast cells, a phosphatidylinositol (Ptdlns) specific 3-kinase encoded by the VPS34 gene has been shown to be required for the sorting of vacuolar/lysosomal hydrolases [23*]. Membrane association of Vps34p and activation of its Ptdlns 3-kinase activity are regulated by a Ser/Thr protein kinase, Vpsl5p, and a model has been proposed whereby this complex regulates vesicle budding and/or cargo loading in the late Golgi [23°,24,25]. In permeabilized rat PC12 cells, a Ptdlns 4-phosphate 5-kinase is required for the priming and exocytosis of secretory granules [26*]. Mammalian cells contain numerous phosphoinositide (PI) 3-kinase isoforms, including a Vps34p homolog [27]; however, the functions of these enzymes are not known. It is clear that different classes of PI 3-kinases, distinguished by their ability to utilize different substrates, can lea6 to distinct cellular responses, suggesting that different signalling pathways may be mediated by different PI products (reviewed in [28",29]). PI 3-kinase activity has been implicated in trafficking events subsequent to receptor internalization, possibly in transport from endosomes to the lysosome. In one set of experiments, tyrosines in the cytoplasmic tail of PDGFR that are phosphorylated in response to P D G F binding were systematically mutated, and the effect of these mutations on P D G F R trafficking was assessed [30]. These mutations interfere with the binding of a variety of Src homology 2 (SH2) domain containing signalling molecules, including the regulatory subunit of PI 3-kinase (p85), the GTPase-activating protein (GAP) of p21Ras, phospholipase C-~' and the phosphotyrosine phosphatase Syp, which all bind activated PDGFR. Specific changes in the tyrosine residues known to bind PI 3-kinase blocked delivery of P D G F R to lysosomes and stabilized the receptor, whereas other mutant receptors trafficked normally [30]. Inhibition of PI 3-kinase activity by the drug wortmannin also blocked lysosomal trafficking of PDGFR and resulted in recycling of the receptor to the plasma membrane, suggesting that PI 3-kinase is required to sort activated P D G F R to the lysosomal delivery pathway [31,32]. Experiments addressing a potential role(s) for PI 3-kinase activity in trafficking of the E G F R have not been reported; however,
552 Membranes and sorting
like PDGFR, the kinase activity of E G F R is required both to sort occupied E G F R to internal vesicles of multivesicular bodies and for subsequent receptor degradation in lysosomes [33,34]. Wortmannin has also been shown to cause mis-sorting of cathepsin D, a lysosomal protease that is sorted from the secretory pathway in the T G N by the mannose 6-phosphate receptor [35,36]. These results imply a role for mammalian PI 3-kinase(s) in endosomal and/or T G N function. T h e observation that PI 3-kinase can modulate Rab5-mediated fusion of endosomes in vitro is consistent with these results [37]. Clearly, localization of the different PI kinase isoforms remains an issue of central importance for understanding the respective roles of the isoforms in mitogenic signalling and regulation of membrane traffic.
Signal-mediated GLUT-4 trafficking One of the most extensively studied signal-transduction pathways is the signalling cascade that results from the binding of insulin to its receptor. Insulin has a dual role: it is both a weak mitogen and a regulator of glucose metabolism. In adipose and muscle tissue, insulin causes a rapid and acute stimulation of glucose uptake. This is mediated primarily by the G L U T - 4 glucose transporter which is sequestered intracellularly within adipocytes and muscle cells and is localized to a portion of the endocytic system. In muscle and fat cells, G L U T - 4
cycles slowly between this endocytic compartment and the plasma membrane, but, upon binding of insulin to the insulin receptor, there is a increase in trafficking from an endocytic 'storage' compartment to the plasma membrane, causing G L U T - 4 to be rapidly translocated to the plasma membrane where it can exert its effect upon glucose uptake (reviewed in [38]; see Fig. 2). T h e signalling pathway that is activated after insulin binds to its receptor is very complex and involves a host of signalling molecules, including the insulin receptor substrate-1 (IRS-1), Grb2-SOS, p21Ras and the p85-p110 form of PI 3-kinase (see [39,40] for reviews). Evidence is emerging to suggest that essentially two divergent signalling pathways result from activation of the insulin receptor and that these pathways separately regulate the mitogenic response and the GLUT-4-trafficking pathway, respectively [41°°,42-44]. After binding insulin, the insulin receptor is rapidly autophosphorylated at multiple tyrosine residues in its cytoplasmic tail domain; these residues provide the binding sites for several SH2 domain containing proteins [45,46"]. Protein-protein interactions mediated by specific phosphotyrosines and SH2 domains form the basis of many of the interactions in this signalling pathway (reviewed in [40]). Although the interactions that are required for
Figure 2 A schematic diagram of some of the membrane trafficking events that occur
either after insulin binding or after activation of Fcy receptors by binding of IgG-coated antigen. Insulin binding to the insulin receptor somehow results in the increased translocation of GLUT-4, via vesicles which bud from an endosomal compartment, to the plasma membrane and in the internalization of the insulin receptor into clathrin-coated pits for subsequent delivery to endosomes and then lysosomes. Phagocytosis of IgG-coated antigen occurs after Fcy-receptor activation which in turn results in a signalling cascade that ultimately causes a rearrangement of the actin cytoskeleton at the site of antigen internalization. Phagocytosed antigen is eventually degraded in structures called phagolysosomes. Common to both these events is receptor phosphorylation. PI 3-kinase has been implicated in many events with the post-TGN and endocytic trafficking pathways, and may function at various points to direct specific proteins into vesicles or facilitate vesicle formation through activation of vesicle coat proteins.
,& *n,uio
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© 1996CurrentOpinionin CellBiology
Receptor signalling and the regulation of endocytic membrane transport Seaman,Burd and Emr 553
the mitogenic effects of insulin are starting to be characterized, components of the machinery required for the translocation of G L U T - 4 to the plasma membrane remain to be formally identified. A strong candidate protein for acting directly in the GLUT-4-translocation pathway is p85-p110 PI 3-kinase. Microinjection of a fusion protein containing the SH2 domain of p85 was able to block both the insulin-stimulated translocation of G L U T - 4 and, consequently, the increase in glucose uptake [41"]. T h e PI 3-kinase inhibitor wortmannin potently blocks the translocation of G L U T - 4 to the plasma membrane [47-49], and dominant-negative mutants of the p85 subunit of PI 3-kinase which fail to bind p l l 0 (the catalytic domain of PI 3-kinase) also block insulin-stimulated glucose uptake but do not block activation of p21Ras [42,49]. Presently, however, the evidence indicates that p85-p110 PI 3-kinase is also involved in the mitogenic pathway [50] and is not exclusively part of the GLUT-4-translocation machinery. This may reflect the fact that PI 3-kinase can catalyze the production of more than one phosphoinositide product (reviewed in [28"]). A simple model therefore is that perhaps PI 3-phosphate (PI 3-P) and/or PI 3,4-bisphosphate (PI 3,4-P 2) is involved in G L U T - 4 translocation, and that PI 3,4,5-trisphosphate (PI 3,4,5-P 3) is required in the mitogenic pathway. Consistent with this model is the finding that PI 3,4,5-P3 can modulate the interaction between phosphotyrosines and SH2 domains ([51"]; there are many more SH2 domain containing proteins believed to be involved in the mitogenic signalling pathway than there are in the GLUT-4-translocation pathway). T h e question remains, however, as to what machinery is involved in the rapid translocation of G L U T - 4 to the plasma membrane. It is now widely accepted that cytoplasmic protein complexes called 'coats' mediate vesicle formation from the compartments that constitute the secretory and endocytic pathways (reviewed in [52,53]). T h e recruitment of coats from the cytoplasm to the membrane involves small GTPases of the ADP-ribosylation factor (ARF) and SAR families. Thus far, there are four distinct coats which have been isolated and characterized, namely clathrin together with either AP1 or AP2, coatomer/COPI, and COPII. Until recently; however, there was no candidate coat protein to mediate the translocation of G L U T - 4 from an endocvtic compartment to the plasma membrane. Recent reports have now provided two possible candidates. One is coatomer/COPI which has recently been implicated in trafficking within the endocytic systcm [54°,55] and which also has a recently established role in the recycling of proteins from the Golgi apparatus to the endoplasmic reticulum [56]. T h e other candidate is clathrin together with an adaptor complex which presumably has yet to be identified. Using immunoelectron microscopy, clathrin was recently localized to a compartment accessible to endocytosed molecules [57°°]. Clathrin is known to act at both the plasma membrane and the T G N to mediate the formation of endocytic vesicles and vesicles required for the delivery
of newly synthesized lysosomal enzymes, respectively; thus, it seems plausible that clathrin may also mediate the formation of GLUT-4-containing vesicles from some part of the endocytic system. A simple hypothesis, therefore, is that upon stimulation by insulin, the budding of vesicles containing G L U T - 4 is upregulated, possibly by stimulation of coat protein recruitment to the endosomal membrane. This ahme may not be sufficient to efficiently deliver G L U T - 4 to the plasma membrane; hence, some form of covalent modification may be required to promotc the loading of G L U T - 4 into vesicles. Presently, however, the mechanism by which the stimulation of coat recruitment could occur has yet to be determined. One possibility is that one of the molecules in the G L U T - 4 ttanslocation signalling pathway acts as a guanine nucleotide exchange factor for an ARF-likc small GTPase. Interestingl3; it has been recently demonstrated that PI 3-kinase can promote GDP for G T P exchange on Rac, another small GTPasc of the p21Ras superfamily [58",59]; this suggests that perhaps PI 3-kinase could act to promote GDP for G T P exchange on an ARF/SAR-Iike small GTPase. This invites speculation as to the role of PI 3-kinases in vesicle coat protein recruitment for GLUT-4-containing vesicles and for other vesicles in the secretory pathway. Although some of the key players in G L U T - 4 translocation are starting to be identified, the process remains something of a 'black box'. Questions regarding the regulation of coat protein assembly/targeting and vesicle formation form a theme throughout the study of membrane traffic, but are particularly pertinent in the case of G L U T - 4 translocation.
Phagocytosis Phagocytosis constitutes another form of ligand uptake from the plasma membrane and is the process by which macrophages and other hematopoietic cells ingest relatively large particles such as bacteria. It differs from endocytosis in that phagocytosis does not employ relatively small vesicles to mediate the internalization process, but instead occurs by large-scale membrane movement which is driven by rearrangement of the actin cytoskeleton close to the plasma membrane. T h e Fcy IgG receptor (FcyR) plays a pivotal role in this process by binding antigens (e.g. bacteria) coated with IgG and initiating a signalling pathway that ultimately leads to rearrangement of the actin cytoskeleton close to the plasma membrane (see Fig. 2). T h e FcyR exists as multiple isoforms; one of the best characterized Fcy receptors is FcyRIIA which is expressed in many hematopoietic cells, including monocvtes neutrophils and B cells (reviewed in [60,61°]). Unlike the insulin receptor and the E G F receptor, the FcyRIIA does not possess intrinsic tyrosine kinase activity that initiates a signalling cascade. Binding of IgG to FcyRIIA results in the phosphorylation of the cytoplasmic domain of the receptor at multiple tyrosine residues which may act as binding sites for SH2 domain
554
Membranes and sorting
containing proteins. Two candidate kinases, p72syk and Fgr, are implicated in the phosphorylation of FcyRIIA [62-64]. Another of the potential targets of these kinases is paxillin, which is phosphorylated after IgG binding to FcyRIIA [63]. Paxillin is a cytoskeleton-associated protein and has therefore been suggested to function in the rearrangement of the actin cytoskeleton that is required for phagocytosis. Also implicated in this role is the protein MARCKS (myristoylated, alanine-rich, C-kinase substrate), which is a substrate for protein kinase C and acts as an actin cross-linking protein [65]. MARCKS associates with nascent phagosomes at the same time as does F-actin. Phosphorylation may regulate the association of MARCKS with phagosomes. As MARCKS is myristoylated at its amino terminus, a modification which allows a protein to associate stably with a membrane, it has been proposed that MARCKS acts in linking actin to the plasma membrane [66"]. Phagocytosis is another cellular process that involves PI 3-kinase activity [67]. p85 is phosphorylated upon FcyR activation, and treatment with wortmannin prevents FcyR-mediated phagocytosis. Presently, however, the role of PI 3-kinase in phagocytosis is not clear.
domain, will play a significant role in the regulatory mechanisms governing membrane traffic. Many important questions clearly remain to be answered and we can anticipate many exciting new developments that will further cement the link between signal transduction and membrane trafficking.
Acknowledgements We thank Sand's" Schmid for critically reading the manuscript. CG Burd is a postdoctoral Fellow of the American Cancer Society. MNJ Seaman is supported by a fellowship from the European Molecular Biology Organisation. SD Emr is an Investigator of the Howard Hughes Medical Institute.
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