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Regulatory Role of SH3 Domain-mediated Protein–Protein Interactions in Synaptic Vesicle Endocytosis
Cell. Signal. Vol. 11, No. 4, pp. 229]238, 1999 Copyright Q 1999 Elsevier Science Inc.
ISSN 0898-6568r99 $ - see front matter PII S0898-6568Ž98.00059-X
TOPICAL REVIEW
Regulatory Role of SH3 Domain-mediated Protein]Protein Interactions in Synaptic Vesicle Endocytosis Peter S. McPhersonU DEPARTMENT
OF
NEUROLOGY AND NEUROSURGERY, MONTREAL NEUROLOGICAL INSTITUTE, MCGILL UNIVERSITY, MONTREAL, PQ H3A 2B4, CANADA
ABSTRACT. Src homology ŽSH. 3 domains are small modules found in a diverse array of proteins. The presence of an SH3 domain confers upon its resident protein the ability to interact with specific proline-rich sequences in protein binding partners. A major focus of research has highlighted a role for SH3 domain-mediated interactions in the regulation of signal transduction events. However, more recent data has suggested an important function for SH3 domains in vesicular trafficking. This review will focus on this newly emerging role with a particular emphasis on the molecular components involved in synaptic vesicle endocytosis and the regulatory role of SH3 domain-mediated protein]protein interactions in this process. CELL SIGNAL 11;4:229]238 Q 1999 Elsevier Science Inc. KEY WORDS. SH3 domain, Endocytosis, Clathrin-coated vesicle, Synaptic vesicle, Dynamin, Amphiphysin, Synaptojanin, Endophilin, EH domain, Eps15, Intersectin, Epsin
INTRODUCTION The accuracy and speed of information processing in the nervous system depends critically on ‘‘fast’’ synaptic transmission. At the presynaptic level, there are two key events underlying this process: the exocytosis of synaptic vesicles ŽSVs. with the release of neurotransmitter, and the endocytosis of SV membranes leading to the restoration of the releasable vesicular pool. The most widely accepted theory suggests that SVs are retrieved by a process of clathrin-mediated endocytosis w1x, which appears to resemble in many respects, clathrin-mediated endocytosis used for a variety of endocytic events in virtually all cells. A model demonstrating some of the key components involved in the formation of clathrin-coated vesicles ŽCCVs. in the nerve terminal is depicted in Fig. 1. Clathrin coats begin to form when the clathrin adaptor protein 2 ŽAP2. is recruited to the plasma membrane where it acts in turn to recruit soluble clathrin triskelia from the cytosol wfor reviews, see w2, 3xx. Several lines of evidence suggest that, in the case of SV endocytosis, synaptotagmin, an integral membrane component of the SV membrane, functions in AP2 recruitment w4]6x. U
Correspondence should be addressed. Tel.: q1 514 3987355; fax: q1 514 3988106; e-mail: [email protected] Received 28 May 1998; accepted 2 July 1998.
Clathrin triskelia assemble into a lattice, and this clathrin scaffold, when attached to the membrane, causes the membrane to deform into a budding vesicle w3x. The clathrin-coated bud continues to grow until it generates an invaginated pit with a narrow neck. Fission of the vesicle neck, in a process mediated by the GTPase dynamin, leads to the formation of a mature CCV. Like clathrin, dynamin is also recruited from a soluble pool in the cytosol to the endocytic site ŽFig. 1. and it is believed that protein]protein interactions mediated by Src homology ŽSH. 3 domains play a critical role in dynamin recruitment. The role of SH3 domain-mediated interactions in several distinct regulatory processes in SV endocytosis will serve as the focus of this review.
SH3 DOMAINS SH3 domains are conserved 50]70 amino acid modules found in a wide variety of proteins w7, 8x. They function by mediating protein]protein interactions with short proline-rich regions in specific ligand partners w9x, and in fact, all proteins that are known to interact with SH3 domains contain at least one copy of the amino acid motif PXXP w10x Žwhere X is any amino acid and invariant prolines ŽP. are underlined.. Screening of phage-displayed and chemically synthesised combinatorial peptide libraries
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FIGURE 1. Diagram illustrating several of the components of clathrin coats that participate in the endocytosis of SVs. Dynamin is modelled as existing in the cytosol as a tetramer [33]. The globular domain in the clathrin triskelia represents the clathrin terminal domain [2, 3, 127] that interacts with the adaptin appendage domain formed by the C-terminus of the adaptin b-subunit [87, 88] (see Fig. 3).
with the SH3 domains of Src and phosphatidylinositol 3-kinase ŽPI3K. have shown that additional residues to this core sequence contribute to specificity w11]14x. These and other studies identified two classes of SH3 ligands, termed class I, with the consensus RXqPXqP and class II, with the consensus qPXqPXR Žwhere q is generally a hydrophobic amino acid, usually proline, leucine, or valine. w11, 12, 15x. Subsequent structural studies of SH3 domain-peptide ligand complexes led to a general model for SH3-ligand interactions w16, 17x. Proline-rich ligands adopt a left-handed polyproline-II helix ŽPPII. conformation in which the two invariant proline residues are found on the same face of the peptide and participate in hydrophobic contacts with conserved aromatic residues in the SH3 domain wdiscussed in w15, 18xx. In the case of class I peptides, an arginine located three amino acids aminoterminal to the PXXP core is often Žbut not always. observed in the high-affinity sites where it can form a salt bridge with a conserved acidic residue found in many SH3 domains. Interestingly, class II peptides also adopt a PPII helix but bind in the opposite orientation to class I peptides Žthus, the arginine located two amino acids carboxy-terminal to the PXXP core interacts with the acidic residue in the SH3 domain.. This general model provides a framework to understanding SH3 domain-ligand interactions but leaves open the question of interaction specificity. More recent analysis of phage-displayed peptide libraries have demonstrated that individual SH3 domains
have distinct specificity’s for potential ligands and can discern subtle differences in the ligand’s primary structure w19, 20x. Much of this specificity appears to come from amino acids that flank the core binding sequences w19, 20x. The presence of SH3 domains in a wide variety of proteins suggests a role for SH3 domain-mediated interactions in a diverse array of cellular processes w18x. However, the best characterised function for SH3 domain-mediated interactions is observed in signalling through receptor tyrosine kinases as seen in the activation of Ras. A central component in the Ras signalling pathway is the growth factor receptor bound protein 2 ŽGrb2., an adaptor protein composed entirely of a central SH2 domain flanked by two SH3 domains w21x. Generally, adaptor proteins are composed entirely of specific protein]protein interaction modules and have no intrinsic enzymatic activity w22x. Thus, Grb2 binds through its SH3 domains to proline-rich sequences in the son of sevenless ŽSos. protein, a guaninenucleotide exchange factors for Ras w23x. Upon autophosphorylation of tyrosine residues in the tails of growth factor receptors, SH2 domain mediated-interactions between Grb2 and the receptor recruit the Grb2rSos complex to the plasma membrane where Sos activates Ras through guanine-nucleotide exchange w23x. Thus, Grb2 serves as an example of an SH3 domain-containing adaptor protein that functions to direct the subcellular localisation of SH3 binding-proteins through protein]protein interactions. A similar mechanism is believed to function
Protein]Protein Interactions in Synaptic Vesicle Endocytosis
in the recruitment of dynamin to the endocytic site in SV endocytosis.
ROLE OF DYNAMIN IN ENDOCYTOSIS An endocytic function for dynamin was first realised based on studies of the temperature-sensitive shibire mutation in Drosophila melanogaster. At the restrictive temperature, shibire flies demonstrate normal SV fusion but vesicle re-internalisation is blocked at the stage of invaginated pits, eventually leading to paralysis w24, 25x. Cloning of the shibire gene product revealed that it encoded the Drosophila homologue of dynamin w26]28x. Expression in mammalian cells of dynamin with a temperature-sensitive mutation comparable to that in shibire w29x, or with mutations in the GTPase domain w30, 31x, also led to a block in endocytosis at the stage of invaginated pits, and as in shibire, the endocytic vesicles were still connected to the plasma membrane via a membranous neck. These studies suggested that dynamin functions late in the endocytic process by mediating the fission of invaginated clathrincoated pits ŽFig. 1.. Confirmation of this idea came from studies using GTPgS that were designed to reconstitute vesicular transport but arrest transport at steps that require GTP hydrolysis w32x. Under these conditions, clathrincoated buds connected to the plasma membrane by narrow tubular necks were often observed. The tubular necks were surrounded by stacks of regularly spaced, electron dense rings formed by dynamin w32x. Independently, it was observed that purified dynamin could spontaneously polymerise into open rings and stacks of rings w33x. Thus, it has been suggested that dynamin rings may function in the budding of CCVs from the plasma membrane by undergoing a GTP-dependent conformational change Ža twist of the ring. that leads to a severing of the vesicle stock. Support for this hypothesis has been provided by the observation that dynamin can act as a GTP-dependent mechanochemical enzyme that causes vesiculation of protein-free liposomes w34, 35x. However, it remains possible that in vivo, dynamin may recruit or activate other effector proteins that function in vesicle budding in a GTP- or ATP-dependent manner w36x. Regardless of the mechanistic details of dynamin function, a central role for the protein in the fission of CCVs from the plasma membrane is well established. Moreover, dynamin has now been implicated in a wide variety of clathrin-mediated endocytic events. For example, dynamin dominant-negative mutants block endocytosis of the EGF receptor w37x as well as agonist-induced endocytosis of G-protein-coupled receptors such as the b2-adrenergic receptor w38x, and specific isoforms of opioid receptors w39, 40x and muscarinic ACh receptors w41, 42x. Dynamin also functions in events as diverse as endocytosis of the glucose transporter GLUT4 w43]46x, the epithelial
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sodium channel w47x, and the neural cell adhesion molecule LI w48x. Although these data support a functional link between dynamin and clathrin-dependent endocytosis in a wide variety of systems, it should be noted that dynamin has also been demonstrated to function in clathrin-independent internalisation of caveolae from the plasma membrane w49, 50x. In addition to an N-terminal GTPase domain, dynamin contains a central pleckstrin homology ŽPH. domain followed by two short predicted coiled-coil domains and a 100 amino acid C-terminal region that is extremely rich in proline residues. One of the first lines of evidence in support of a role for SH3 domains in vesicular trafficking came with the observation that the SH3 domains of Grb2, PI3K, and phospholipase C g bind to the proline-rich C-terminus of dynamin in vitro w51]56x. In fact, this region contains at least three distinct SH3 domain-binding sites, each with a different SH3 domain-binding specificity w57, 58x. One important consequence of SH3 domain-binding is stimulation of dynamin’s GTPase activity w51, 52x. Thus, it is interesting to note that a number of SH3 domain-containing proteins that interact with dynamin also contain SH2 domains. This may allow for activation of dynamin at sites of growth factor receptor endocytosis as appears to be the case for EGF receptor endocytosis which requires Grb2 mediated recruitment of dynamin to the receptor w59x.
THE SH3 DOMAIN-CONTAINING PROTEINS AMPHIPHYSIN I AND II FUNCTION IN SV ENDOCYTOSIS Of the various SH3 domain-containing proteins that interact with dynamin, amphiphysin I has been best characterised as having an important regulatory role in dynamin function in vivo. Amphiphysin I, which was originally isolated from chicken synaptic fractions w60x, has an SH3 domain at its C-terminus w61x ŽFig. 2. which interacts with high affinity and specificity to a single class II SH3 domain-binding site ŽPSRPNR. in the proline-rich Cterminus of dynamin w57, 58, 62x. Like dynamin, amphiphysin I is enriched in nerve terminals w62x where it is found on clathrin-coated membranes and dynamin-coated membrane tubules following GTPgS treatment w63x, and the two proteins can be co-immunoprecipitated from brain extracts w62x. Injection of the SH3 domain of amphiphysin I into the lamprey giant reticulospinal synapse causes a block in SV endocytosis at a stage of invaginated clathrin-coated pits which remain connected to the plasma membrane by a narrow membrane neck w64x. However, the vesicle necks are not coated with dynamin rings as is seen in shibire flies suggesting that the block in endocytosis is due to a block in dynamin recruitment to the endocytic site w64x. Further, over-expression of the am-
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FIGURE 2. Domain model of amphiphysins [adapted from ref. [75]). The figure shows amphiphysin II splice variants isolated from human cortex cDNA (type IIa ]IId) [75] compared to the splice variant SH3P9 [66] and with amphiphysin I, which is the product of a distinct gene [60, 61]. The numbers in bold above amphiphysin IIa represent the amino acid numbers of some intron-exon boundaries [70, 74] and the numbers in italics represent exons as defined by Wechsler-Reya et al. [74]. Although the N-terminal insert domain was identified in cDNA clones from several different laboratories [68 ]72], an exon encoding this region was not identified in the amphiphysin II gene [74].
phiphysin I SH3 domain in COS cells leads to a block in both transferrin receptor and EGF receptor endocytosis w65x and injection of the SH3 domain of amphiphysin I into 3T3-L1 adipocytes blocks endocytosis of GLUT4 vesicles w44x. Taken together, these results suggest that a specific interaction between the SH3 domain of amphiphysin I and dynamin plays a critical role in endocytic function. Another important SH3 domain-containing protein that appears to function in endocytosis in the nerve terminal is amphiphysin II. Amphiphysin II was identified in the form of multiple splice variants in several laboratories w66]73x. Analysis of the gene encoding amphiphysin II demonstrates that it is composed of nineteen exons w74x ŽFigs. 2 and 3.. Of interest, four of the exons Ž12a]12d. are expressed specifically in the brain w74x and their alternative usage leads, in part, to the generation of the multiple amphiphysin II splice variants ŽType IIa, IIb, IIc, and IId. identified by PCR from human cortex cDNA w75x ŽFig. 2.. Like amphiphysin I, the major brain form of amphiphysin II Žapparently corresponding to amphiphysin IIa. is concentrated in nerve terminals w70, 72x where it interacts through its SH3 domain with dynamin w68, 70, 72x. In
fact, amphiphysin I and II appear to form heterodimers w72x and a stable amphiphysin IrII complex with dynamin can be immunoprecipitated from rat brain synaptic fractions w76, 77x. It is likely that the amphiphysin IrII-dynamin complex functions in targeting dynamin to the site of SV endocytosis ŽFig. 4.. An important role for SH3 domains in directing subcellular targeting was first suggested by studies in which SH3 domain-dependent interactions were seen to direct signalling proteins to specific subcellular locations after injection into fibroblasts w78x. In fact, the major function for Grb2 in the Ras signalling pathway is to direct Sos to the plasma membrane where it can catalyse the guanine-nucleotide exchange of Ras w23, 79, 80x. Dynamin also appears to be targeted to the plasma membrane in an SH3 domain-dependent manner as removal of a 9-amino acid SH3 domain-binding site in dynamin’s proline-rich tail prevents its targeting to clathrin-containing domains on the plasma membrane w81x. Furthermore, blocking the SH3 domain-dependent interaction between dynamin and amphiphysin IrII disrupts endocytosis, likely by preventing dynamin targeting to the endocytic site w64x, although the SH3 domain of amphiphysin II may
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FIGURE 3. Clathrin-binding domains in amphiphysin II. The sequence of exons 12b and 12c from amphiphysin II (see Fig. 2) is shown aligned to a corresponding region from amphiphysin I, regions of adaptin b subunits, arrestin3 and b-arrestin. Two separate clathrin-binding domains, with the sequences LLDLDFDP and PWDLW, identified in amphiphysin II, are conserved in amphiphysin I [75]. Of interest, the sequence LLDLD is similar to clathrin-binding sequences found in the other proteins and helps define a clathrin-binding domain with the consensus sequence L(L,I)(D,E,N)(L,F)(D,E) (see also ref. [87]).
also block endocytosis by preventing dynamin oligomerization w82x. Subcellular fractionation of rat brain extracts reveals that the amphiphysins are predominantly soluble proteins although a fraction of each protein is membrane associated w70, 72x. This suggests that amphiphysins may function in an analogous manner to Grb2 by remaining in a stable association with dynamin in the cytosol and mediating the transfer of dynamin to the plasma mem-
brane to catalyse CCV formation. Thus, there is a requirement for an SH3 domain-independent siteŽs. in the amphiphysins that could mediate their transient interaction with emergent clathrin-coated buds. In fact, the amphiphysins interact with two distinct binding partners that likely serve this targeting function. First, amphiphysin I has been demonstrated to bind to the multisubunit clathrin adaptor protein AP2 through
FIGURE 4. Potential role for the amphiphysin targeting complex in endocytosis. (PRD =proline-rich domain).
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the C-terminal appendage domain of the a-adaptin subunit w62, 77, 83x ŽFig. 4.. In addition, both amphiphysin I and II bind directly to clathrin w70, 77, 84x. In fact, amphiphysin II contains two distinct and independent clathrin-binding sites, one of five ŽPWDLW. and the other of nine ŽLLDLDFDP. amino acids, both of which are conserved in amphiphysin I ŽFig. 3. w75x. Interestingly, these two clathrin-binding domains are encoded by separate exons Žexons 12b and 12c; Figs. 2 and 3. that are alternatively used in a brain specific manner w74x. One of the sites, with the sequence LLDLDFDP, strongly resembles a site in b-arrestin ŽLIEFE. Žnote the presence of alternating hydrophobic and acidic residues. that is critical for b-arrestin binding to clathrin w85x. Furthermore, this site is similar to the clathrin-binding site recently identified in the b3 subunit of the newly identified clathrin adaptor AP3 w86, 87x and a similar site is seen in the clathrin-binding region of the b1 and b2 subunits from the clathrin adaptors AP1 and AP2, respectively w88x. When aligned, these sequences define a novel clathrin-binding domain with the sequence LŽL,I.ŽD,E,N.ŽL,F.ŽD,E. w87x ŽFig. 3.. Thus, through interactions with clathrin and AP2, the amphiphysin IrII-dynamin complex could be targeted to endocytic sites with the amphiphysins functioning as molecular adaptors. Of interest, it appears that the formation and function of this complex is regulated by the phosphorylation state of its components w77, 89x. Thus, Ca2q influx during nerve-terminal depolarisation activates the Ca2q-dependent phosphatase, calcineurin, which rapidly dephosphorylates dynamin, amphiphysin I and II, and synaptojanin w77, 89x. Dephosphorylation of dynamin and synaptojanin increases their binding to the amphiphysins, and dephosphorylation of the amphiphysins increases their binding to clathrin and AP2 w77x. These data suggest an attractive mechanism by which Ca2q influx during nerve terminal depolarization-dependent SV exocytosis could lead to the assembly of the endocytic machinery necessary for SV endocytosis w77x.
SYNAPTOJANIN, AN INOSITOL 5-PHOSPHATASE IN MEMBRANE TRAFFICKING Another component of the amphiphysin IrII-dynamin targeting complex is synaptojanin w76, 77x ŽFig. 4.. Synaptojanin was originally isolated as a major SH3 domain-binding protein in the brain w55x and its cloning revealed a unique three domain structure w90x. The N-terminus is homologous to the yeast protein, Sac1, which has been genetically implicated in the regulation of phospholipid metabolism w91x. The central region reveals synaptojanin
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to be a member of the 5-phosphatase family of enzymes and synaptojanin has 5-phosphatase activity directed towards a variety of soluble inositol polyphosphates and inositol phospholipids w90, 92]94x. Phosphoinositides function in regulating a number of important proteins involved in synaptic vesicle endocytosis including the clathrin adaptors AP2 and AP180 w95, 96x, dynamin w97]99x, and synaptotagmin w6x. The role of phosphoinositides in the regulation of synaptic vesicle endocytosis as well as a variety of other vesicular trafficking events has been the subject of a recent review w91x. The C-terminus of synaptojanin, which is proline-rich, contains several SH3 domain consensus-binding sites and in fact, amphiphysin I and II interact in vitro with synaptojanin through distinct SH3 binding sequences w76x. Synaptojanin is highly enriched in brain and is concentrated in the presynaptic nerve terminal w100x where, like dynamin and amphiphysin, it is found in part on clathrin-coated endocytic structures w101x implicating a functional role for synaptojanin in endocytosis in the nerve terminal. As synaptojanin is a component of the amphiphysin IrII endocytic targeting complex, it appears likely that amphiphysin I and II function to target synaptojanin as well as dynamin to the endocytic site. In order to identify novel proteins potentially involved in regulating synaptojanin function, we used a biochemical screen to identify brain proteins that interact directly with synaptojanin. In addition to amphiphysin I and II, we identified a 40 kDa protein Žtermed endophilin. as the major synaptojanin-binding protein in the brain w102x. Partial purification of the protein, followed by peptide sequence analysis, revealed it to be identical to SH3P4, a protein that was identified as a member of a novel family of SH3 domain-containing proteins Žalong with SH3P8 and SH3P13. through a screen of an expression library with a Src class II SH3 domain peptide ligand w66x. SH3P4rendophilin was simultaneously identified as a major synaptojanin-binding protein through a yeast two-hybrid screen with the proline-rich C-terminus of synaptojanin w103x. Endophilin binds through its SH3 domain to a site in the proline-rich C-terminus of synaptojanin which is distinct from the sites for amphiphysin I and II w76x. Endophilin is brain specific and is highly concentrated in the presynaptic nerve terminal where it associates in a stable complex with synaptojanin in vivo w76, 103x. The functional role of the synaptojaninrendophilin complex is currently unknown. However, it may function to regulate the amount of synaptojanin at the endocytic site by regulating its accessibility for the amphiphysin IrII endocytic targeting complex. Alternatively, endophilin may not simply buffer excess synaptojanin, but may actively target it to other sites within the neuron through its central domain which has a high probability of participating in coiled-coil interactions.
Protein]Protein Interactions in Synaptic Vesicle Endocytosis
EH DOMAINS IN ENDOCYTOSIS: EVIDENCE FOR AN EH DOMAIN-SH3 DOMAIN CONNECTION Recently, attention has focused on a new protein]protein interaction domain that appears to play a role in endocytosis. This 60]70 amino acid module was first recognised in the protein Eps15 and has been referred to as the Eps15 homology ŽEH. domain w104x. Eps15, which contains three copies of the EH domain at its N-terminus, is constitutively bound through its C-terminus to the aadaptin subunit of AP2 w105]107x. Eps15-AP2 interactions are regulated by clathrin assembly w108x and Eps15 has been localised to the rim of clathrin-coated pits w109x. In Saccharomyces cerevisiae, End3p and Pan1p are required for endocytosis and contain one and two copies of the EH domain, respectively w110]113x. The EH domain appears to function as a protein]protein interaction module w104x and the amino acids asparagine-proline-phenylalanine ŽNPF. form the core of an EH domain-binding motif w114]117x. An interesting connection between EH domains and SH3 domains has now arisen from two independent observations. First, it has been demonstrated that synaptojanin-170, an alternatively spliced form of synaptojanin with a broad tissue distribution w90, 118x, binds to the EH domains of Eps15 in vitro w101x. Synaptojanin-170 has an identical domain structure as synaptojanin Žincluding the proline-rich sites that interact with SH3 domain-containing proteins. but contains an additional 25 kDa C-terminal region with three NPF repeats that mediate the EH domain interaction w101, 119x. It is interesting to note that the EH domain-containing yeast PAN1 gene interacts genetically with SJL1 w120x, one of three synaptojanin-like proteins in yeast w121x. A second link between EH domains and SH3 domains has come with the identification of intersectin, a protein composed of two N-terminal EH domains, a central helix forming region, and five C-terminal SH3 domains w117, 122x. The EH domains of intersectin also interact with the NPF core motif and interestingly, a subset of the SH3 domains interact with dynamin w117x. Intersectin is homologous with Dap160, a Drosophila protein with two EH domains and four SH3 domains that was identified based on its affinity for dynamin w123x. Screening of a mouse expression library with the intersectin EH domains has identified two novel intersectin-binding proteins ŽIbps. w117x. The Ibps are the mouse homolgues of rat epsin, a protein identified based on its interactions with the EH domains of Eps15 w124x. Epsin appears to be localised to clathrin-coated pits in situ, likely through its binding to the coat protein AP2 w124x. In addition to the presence of AP2-binding sites and multiple copies of the NPF motif, it is intriguing to note that the Ibpsrepsin contain the consensus clathrin-binding domain previously discussed. In fact, a protein complex containing intersectin with
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dynamin, AP2 and clathrin can be isolated from rat brain Žour unpublished results.. Thus, it appears that intersectin may function as an adaptor protein in endocytosis by bringing together novel clathrinrEH domain-binding proteins with SH3 domain-binding proteins such as dynamin.
CONCLUSIONS Both dynamin and synaptojanin, which have been localised to endocytic intermediates in the nerve terminal, appear to play critical roles in the formation of CCVs from the plasma membrane. As both are soluble proteins present primarily in the cytoplasm, it is important to understand the mechanisms that allow them to transiently localise to endocytic sites. In several different systems, SH3 domain-containing adaptor proteins function by localising their appropriate SH3 domain-binding partner to a specific subcellular localisation Žsuch as the plasma membrane. w22x. Recent molecular and biochemical data has identified several SH3 domain-containing proteins that appear to function as molecular adaptors in endocytosis. Thus, amphiphysin I and II, which interact through their SH3 domains with dynamin and synaptojanin, likely function by targeting these two key enzymes to endocytic domains through non-SH3 domain interactions with AP2 and clathrin. Endophilin may function by regulating this targeting complex or by mediating the targeting of synaptojanin and dynamin to distinct membrane or cytoskeletal sites. Intersectin may be involved in organising SH3 domain- and EH domain-binding proteins at the endocytic site. Research over the last several years has begun to reveal an unanticipated molecular complexity in clathrin-mediated endocytosis. This appears to be in contrast to some other vesicular budding events, such as the formation of COPII vesicles from the endoplasmic reticulum, which appears to require relatively few protein components w125x. Perhaps this complexity reflects the multiple roles for clathrin-mediated vesicular budding. Whereas COPII vesicles are used at a specific stage of vesicular trafficking w125x, clathrin-coated vesicles are used for budding from the TGN as well as for endocytosis from the plasma membrane. Further, in addition to synaptic vesicle membranes, a diverse array of proteins including receptors, ion channels, and membrane transporters undergo clathrinmediated endocytosis in both a regulated and constitutive manner. Complexity may also arise from the high demand for speed and efficiency placed on the endocytic machinery by SV endocytosis. Thus, neuronal cells express most endocytic proteins at much higher levels than non-neuronal cells and often express neuron specific isoforms w126x. To fully understand clathrin-mediated endocytosis and its role in the endocytosis of SVs, it will be critical to identify
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the full complement of proteins functioning in this process at which time we can begin to develop more precise molecular models which will eventually lead to a more complete understanding of this complex and fundamental event.
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ISSN 0898-6568r99 $ - see front matter PII S0898-6568Ž98.00059-X
TOPICAL REVIEW
Regulatory Role of SH3 Domain-mediated Protein]Protein Interactions in Synaptic Vesicle Endocytosis Peter S. McPhersonU DEPARTMENT
OF
NEUROLOGY AND NEUROSURGERY, MONTREAL NEUROLOGICAL INSTITUTE, MCGILL UNIVERSITY, MONTREAL, PQ H3A 2B4, CANADA
ABSTRACT. Src homology ŽSH. 3 domains are small modules found in a diverse array of proteins. The presence of an SH3 domain confers upon its resident protein the ability to interact with specific proline-rich sequences in protein binding partners. A major focus of research has highlighted a role for SH3 domain-mediated interactions in the regulation of signal transduction events. However, more recent data has suggested an important function for SH3 domains in vesicular trafficking. This review will focus on this newly emerging role with a particular emphasis on the molecular components involved in synaptic vesicle endocytosis and the regulatory role of SH3 domain-mediated protein]protein interactions in this process. CELL SIGNAL 11;4:229]238 Q 1999 Elsevier Science Inc. KEY WORDS. SH3 domain, Endocytosis, Clathrin-coated vesicle, Synaptic vesicle, Dynamin, Amphiphysin, Synaptojanin, Endophilin, EH domain, Eps15, Intersectin, Epsin
INTRODUCTION The accuracy and speed of information processing in the nervous system depends critically on ‘‘fast’’ synaptic transmission. At the presynaptic level, there are two key events underlying this process: the exocytosis of synaptic vesicles ŽSVs. with the release of neurotransmitter, and the endocytosis of SV membranes leading to the restoration of the releasable vesicular pool. The most widely accepted theory suggests that SVs are retrieved by a process of clathrin-mediated endocytosis w1x, which appears to resemble in many respects, clathrin-mediated endocytosis used for a variety of endocytic events in virtually all cells. A model demonstrating some of the key components involved in the formation of clathrin-coated vesicles ŽCCVs. in the nerve terminal is depicted in Fig. 1. Clathrin coats begin to form when the clathrin adaptor protein 2 ŽAP2. is recruited to the plasma membrane where it acts in turn to recruit soluble clathrin triskelia from the cytosol wfor reviews, see w2, 3xx. Several lines of evidence suggest that, in the case of SV endocytosis, synaptotagmin, an integral membrane component of the SV membrane, functions in AP2 recruitment w4]6x. U
Correspondence should be addressed. Tel.: q1 514 3987355; fax: q1 514 3988106; e-mail: [email protected] Received 28 May 1998; accepted 2 July 1998.
Clathrin triskelia assemble into a lattice, and this clathrin scaffold, when attached to the membrane, causes the membrane to deform into a budding vesicle w3x. The clathrin-coated bud continues to grow until it generates an invaginated pit with a narrow neck. Fission of the vesicle neck, in a process mediated by the GTPase dynamin, leads to the formation of a mature CCV. Like clathrin, dynamin is also recruited from a soluble pool in the cytosol to the endocytic site ŽFig. 1. and it is believed that protein]protein interactions mediated by Src homology ŽSH. 3 domains play a critical role in dynamin recruitment. The role of SH3 domain-mediated interactions in several distinct regulatory processes in SV endocytosis will serve as the focus of this review.
SH3 DOMAINS SH3 domains are conserved 50]70 amino acid modules found in a wide variety of proteins w7, 8x. They function by mediating protein]protein interactions with short proline-rich regions in specific ligand partners w9x, and in fact, all proteins that are known to interact with SH3 domains contain at least one copy of the amino acid motif PXXP w10x Žwhere X is any amino acid and invariant prolines ŽP. are underlined.. Screening of phage-displayed and chemically synthesised combinatorial peptide libraries
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FIGURE 1. Diagram illustrating several of the components of clathrin coats that participate in the endocytosis of SVs. Dynamin is modelled as existing in the cytosol as a tetramer [33]. The globular domain in the clathrin triskelia represents the clathrin terminal domain [2, 3, 127] that interacts with the adaptin appendage domain formed by the C-terminus of the adaptin b-subunit [87, 88] (see Fig. 3).
with the SH3 domains of Src and phosphatidylinositol 3-kinase ŽPI3K. have shown that additional residues to this core sequence contribute to specificity w11]14x. These and other studies identified two classes of SH3 ligands, termed class I, with the consensus RXqPXqP and class II, with the consensus qPXqPXR Žwhere q is generally a hydrophobic amino acid, usually proline, leucine, or valine. w11, 12, 15x. Subsequent structural studies of SH3 domain-peptide ligand complexes led to a general model for SH3-ligand interactions w16, 17x. Proline-rich ligands adopt a left-handed polyproline-II helix ŽPPII. conformation in which the two invariant proline residues are found on the same face of the peptide and participate in hydrophobic contacts with conserved aromatic residues in the SH3 domain wdiscussed in w15, 18xx. In the case of class I peptides, an arginine located three amino acids aminoterminal to the PXXP core is often Žbut not always. observed in the high-affinity sites where it can form a salt bridge with a conserved acidic residue found in many SH3 domains. Interestingly, class II peptides also adopt a PPII helix but bind in the opposite orientation to class I peptides Žthus, the arginine located two amino acids carboxy-terminal to the PXXP core interacts with the acidic residue in the SH3 domain.. This general model provides a framework to understanding SH3 domain-ligand interactions but leaves open the question of interaction specificity. More recent analysis of phage-displayed peptide libraries have demonstrated that individual SH3 domains
have distinct specificity’s for potential ligands and can discern subtle differences in the ligand’s primary structure w19, 20x. Much of this specificity appears to come from amino acids that flank the core binding sequences w19, 20x. The presence of SH3 domains in a wide variety of proteins suggests a role for SH3 domain-mediated interactions in a diverse array of cellular processes w18x. However, the best characterised function for SH3 domain-mediated interactions is observed in signalling through receptor tyrosine kinases as seen in the activation of Ras. A central component in the Ras signalling pathway is the growth factor receptor bound protein 2 ŽGrb2., an adaptor protein composed entirely of a central SH2 domain flanked by two SH3 domains w21x. Generally, adaptor proteins are composed entirely of specific protein]protein interaction modules and have no intrinsic enzymatic activity w22x. Thus, Grb2 binds through its SH3 domains to proline-rich sequences in the son of sevenless ŽSos. protein, a guaninenucleotide exchange factors for Ras w23x. Upon autophosphorylation of tyrosine residues in the tails of growth factor receptors, SH2 domain mediated-interactions between Grb2 and the receptor recruit the Grb2rSos complex to the plasma membrane where Sos activates Ras through guanine-nucleotide exchange w23x. Thus, Grb2 serves as an example of an SH3 domain-containing adaptor protein that functions to direct the subcellular localisation of SH3 binding-proteins through protein]protein interactions. A similar mechanism is believed to function
Protein]Protein Interactions in Synaptic Vesicle Endocytosis
in the recruitment of dynamin to the endocytic site in SV endocytosis.
ROLE OF DYNAMIN IN ENDOCYTOSIS An endocytic function for dynamin was first realised based on studies of the temperature-sensitive shibire mutation in Drosophila melanogaster. At the restrictive temperature, shibire flies demonstrate normal SV fusion but vesicle re-internalisation is blocked at the stage of invaginated pits, eventually leading to paralysis w24, 25x. Cloning of the shibire gene product revealed that it encoded the Drosophila homologue of dynamin w26]28x. Expression in mammalian cells of dynamin with a temperature-sensitive mutation comparable to that in shibire w29x, or with mutations in the GTPase domain w30, 31x, also led to a block in endocytosis at the stage of invaginated pits, and as in shibire, the endocytic vesicles were still connected to the plasma membrane via a membranous neck. These studies suggested that dynamin functions late in the endocytic process by mediating the fission of invaginated clathrincoated pits ŽFig. 1.. Confirmation of this idea came from studies using GTPgS that were designed to reconstitute vesicular transport but arrest transport at steps that require GTP hydrolysis w32x. Under these conditions, clathrincoated buds connected to the plasma membrane by narrow tubular necks were often observed. The tubular necks were surrounded by stacks of regularly spaced, electron dense rings formed by dynamin w32x. Independently, it was observed that purified dynamin could spontaneously polymerise into open rings and stacks of rings w33x. Thus, it has been suggested that dynamin rings may function in the budding of CCVs from the plasma membrane by undergoing a GTP-dependent conformational change Ža twist of the ring. that leads to a severing of the vesicle stock. Support for this hypothesis has been provided by the observation that dynamin can act as a GTP-dependent mechanochemical enzyme that causes vesiculation of protein-free liposomes w34, 35x. However, it remains possible that in vivo, dynamin may recruit or activate other effector proteins that function in vesicle budding in a GTP- or ATP-dependent manner w36x. Regardless of the mechanistic details of dynamin function, a central role for the protein in the fission of CCVs from the plasma membrane is well established. Moreover, dynamin has now been implicated in a wide variety of clathrin-mediated endocytic events. For example, dynamin dominant-negative mutants block endocytosis of the EGF receptor w37x as well as agonist-induced endocytosis of G-protein-coupled receptors such as the b2-adrenergic receptor w38x, and specific isoforms of opioid receptors w39, 40x and muscarinic ACh receptors w41, 42x. Dynamin also functions in events as diverse as endocytosis of the glucose transporter GLUT4 w43]46x, the epithelial
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sodium channel w47x, and the neural cell adhesion molecule LI w48x. Although these data support a functional link between dynamin and clathrin-dependent endocytosis in a wide variety of systems, it should be noted that dynamin has also been demonstrated to function in clathrin-independent internalisation of caveolae from the plasma membrane w49, 50x. In addition to an N-terminal GTPase domain, dynamin contains a central pleckstrin homology ŽPH. domain followed by two short predicted coiled-coil domains and a 100 amino acid C-terminal region that is extremely rich in proline residues. One of the first lines of evidence in support of a role for SH3 domains in vesicular trafficking came with the observation that the SH3 domains of Grb2, PI3K, and phospholipase C g bind to the proline-rich C-terminus of dynamin in vitro w51]56x. In fact, this region contains at least three distinct SH3 domain-binding sites, each with a different SH3 domain-binding specificity w57, 58x. One important consequence of SH3 domain-binding is stimulation of dynamin’s GTPase activity w51, 52x. Thus, it is interesting to note that a number of SH3 domain-containing proteins that interact with dynamin also contain SH2 domains. This may allow for activation of dynamin at sites of growth factor receptor endocytosis as appears to be the case for EGF receptor endocytosis which requires Grb2 mediated recruitment of dynamin to the receptor w59x.
THE SH3 DOMAIN-CONTAINING PROTEINS AMPHIPHYSIN I AND II FUNCTION IN SV ENDOCYTOSIS Of the various SH3 domain-containing proteins that interact with dynamin, amphiphysin I has been best characterised as having an important regulatory role in dynamin function in vivo. Amphiphysin I, which was originally isolated from chicken synaptic fractions w60x, has an SH3 domain at its C-terminus w61x ŽFig. 2. which interacts with high affinity and specificity to a single class II SH3 domain-binding site ŽPSRPNR. in the proline-rich Cterminus of dynamin w57, 58, 62x. Like dynamin, amphiphysin I is enriched in nerve terminals w62x where it is found on clathrin-coated membranes and dynamin-coated membrane tubules following GTPgS treatment w63x, and the two proteins can be co-immunoprecipitated from brain extracts w62x. Injection of the SH3 domain of amphiphysin I into the lamprey giant reticulospinal synapse causes a block in SV endocytosis at a stage of invaginated clathrin-coated pits which remain connected to the plasma membrane by a narrow membrane neck w64x. However, the vesicle necks are not coated with dynamin rings as is seen in shibire flies suggesting that the block in endocytosis is due to a block in dynamin recruitment to the endocytic site w64x. Further, over-expression of the am-
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FIGURE 2. Domain model of amphiphysins [adapted from ref. [75]). The figure shows amphiphysin II splice variants isolated from human cortex cDNA (type IIa ]IId) [75] compared to the splice variant SH3P9 [66] and with amphiphysin I, which is the product of a distinct gene [60, 61]. The numbers in bold above amphiphysin IIa represent the amino acid numbers of some intron-exon boundaries [70, 74] and the numbers in italics represent exons as defined by Wechsler-Reya et al. [74]. Although the N-terminal insert domain was identified in cDNA clones from several different laboratories [68 ]72], an exon encoding this region was not identified in the amphiphysin II gene [74].
phiphysin I SH3 domain in COS cells leads to a block in both transferrin receptor and EGF receptor endocytosis w65x and injection of the SH3 domain of amphiphysin I into 3T3-L1 adipocytes blocks endocytosis of GLUT4 vesicles w44x. Taken together, these results suggest that a specific interaction between the SH3 domain of amphiphysin I and dynamin plays a critical role in endocytic function. Another important SH3 domain-containing protein that appears to function in endocytosis in the nerve terminal is amphiphysin II. Amphiphysin II was identified in the form of multiple splice variants in several laboratories w66]73x. Analysis of the gene encoding amphiphysin II demonstrates that it is composed of nineteen exons w74x ŽFigs. 2 and 3.. Of interest, four of the exons Ž12a]12d. are expressed specifically in the brain w74x and their alternative usage leads, in part, to the generation of the multiple amphiphysin II splice variants ŽType IIa, IIb, IIc, and IId. identified by PCR from human cortex cDNA w75x ŽFig. 2.. Like amphiphysin I, the major brain form of amphiphysin II Žapparently corresponding to amphiphysin IIa. is concentrated in nerve terminals w70, 72x where it interacts through its SH3 domain with dynamin w68, 70, 72x. In
fact, amphiphysin I and II appear to form heterodimers w72x and a stable amphiphysin IrII complex with dynamin can be immunoprecipitated from rat brain synaptic fractions w76, 77x. It is likely that the amphiphysin IrII-dynamin complex functions in targeting dynamin to the site of SV endocytosis ŽFig. 4.. An important role for SH3 domains in directing subcellular targeting was first suggested by studies in which SH3 domain-dependent interactions were seen to direct signalling proteins to specific subcellular locations after injection into fibroblasts w78x. In fact, the major function for Grb2 in the Ras signalling pathway is to direct Sos to the plasma membrane where it can catalyse the guanine-nucleotide exchange of Ras w23, 79, 80x. Dynamin also appears to be targeted to the plasma membrane in an SH3 domain-dependent manner as removal of a 9-amino acid SH3 domain-binding site in dynamin’s proline-rich tail prevents its targeting to clathrin-containing domains on the plasma membrane w81x. Furthermore, blocking the SH3 domain-dependent interaction between dynamin and amphiphysin IrII disrupts endocytosis, likely by preventing dynamin targeting to the endocytic site w64x, although the SH3 domain of amphiphysin II may
Protein]Protein Interactions in Synaptic Vesicle Endocytosis
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FIGURE 3. Clathrin-binding domains in amphiphysin II. The sequence of exons 12b and 12c from amphiphysin II (see Fig. 2) is shown aligned to a corresponding region from amphiphysin I, regions of adaptin b subunits, arrestin3 and b-arrestin. Two separate clathrin-binding domains, with the sequences LLDLDFDP and PWDLW, identified in amphiphysin II, are conserved in amphiphysin I [75]. Of interest, the sequence LLDLD is similar to clathrin-binding sequences found in the other proteins and helps define a clathrin-binding domain with the consensus sequence L(L,I)(D,E,N)(L,F)(D,E) (see also ref. [87]).
also block endocytosis by preventing dynamin oligomerization w82x. Subcellular fractionation of rat brain extracts reveals that the amphiphysins are predominantly soluble proteins although a fraction of each protein is membrane associated w70, 72x. This suggests that amphiphysins may function in an analogous manner to Grb2 by remaining in a stable association with dynamin in the cytosol and mediating the transfer of dynamin to the plasma mem-
brane to catalyse CCV formation. Thus, there is a requirement for an SH3 domain-independent siteŽs. in the amphiphysins that could mediate their transient interaction with emergent clathrin-coated buds. In fact, the amphiphysins interact with two distinct binding partners that likely serve this targeting function. First, amphiphysin I has been demonstrated to bind to the multisubunit clathrin adaptor protein AP2 through
FIGURE 4. Potential role for the amphiphysin targeting complex in endocytosis. (PRD =proline-rich domain).
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the C-terminal appendage domain of the a-adaptin subunit w62, 77, 83x ŽFig. 4.. In addition, both amphiphysin I and II bind directly to clathrin w70, 77, 84x. In fact, amphiphysin II contains two distinct and independent clathrin-binding sites, one of five ŽPWDLW. and the other of nine ŽLLDLDFDP. amino acids, both of which are conserved in amphiphysin I ŽFig. 3. w75x. Interestingly, these two clathrin-binding domains are encoded by separate exons Žexons 12b and 12c; Figs. 2 and 3. that are alternatively used in a brain specific manner w74x. One of the sites, with the sequence LLDLDFDP, strongly resembles a site in b-arrestin ŽLIEFE. Žnote the presence of alternating hydrophobic and acidic residues. that is critical for b-arrestin binding to clathrin w85x. Furthermore, this site is similar to the clathrin-binding site recently identified in the b3 subunit of the newly identified clathrin adaptor AP3 w86, 87x and a similar site is seen in the clathrin-binding region of the b1 and b2 subunits from the clathrin adaptors AP1 and AP2, respectively w88x. When aligned, these sequences define a novel clathrin-binding domain with the sequence LŽL,I.ŽD,E,N.ŽL,F.ŽD,E. w87x ŽFig. 3.. Thus, through interactions with clathrin and AP2, the amphiphysin IrII-dynamin complex could be targeted to endocytic sites with the amphiphysins functioning as molecular adaptors. Of interest, it appears that the formation and function of this complex is regulated by the phosphorylation state of its components w77, 89x. Thus, Ca2q influx during nerve-terminal depolarisation activates the Ca2q-dependent phosphatase, calcineurin, which rapidly dephosphorylates dynamin, amphiphysin I and II, and synaptojanin w77, 89x. Dephosphorylation of dynamin and synaptojanin increases their binding to the amphiphysins, and dephosphorylation of the amphiphysins increases their binding to clathrin and AP2 w77x. These data suggest an attractive mechanism by which Ca2q influx during nerve terminal depolarization-dependent SV exocytosis could lead to the assembly of the endocytic machinery necessary for SV endocytosis w77x.
SYNAPTOJANIN, AN INOSITOL 5-PHOSPHATASE IN MEMBRANE TRAFFICKING Another component of the amphiphysin IrII-dynamin targeting complex is synaptojanin w76, 77x ŽFig. 4.. Synaptojanin was originally isolated as a major SH3 domain-binding protein in the brain w55x and its cloning revealed a unique three domain structure w90x. The N-terminus is homologous to the yeast protein, Sac1, which has been genetically implicated in the regulation of phospholipid metabolism w91x. The central region reveals synaptojanin
P. S. McPherson
to be a member of the 5-phosphatase family of enzymes and synaptojanin has 5-phosphatase activity directed towards a variety of soluble inositol polyphosphates and inositol phospholipids w90, 92]94x. Phosphoinositides function in regulating a number of important proteins involved in synaptic vesicle endocytosis including the clathrin adaptors AP2 and AP180 w95, 96x, dynamin w97]99x, and synaptotagmin w6x. The role of phosphoinositides in the regulation of synaptic vesicle endocytosis as well as a variety of other vesicular trafficking events has been the subject of a recent review w91x. The C-terminus of synaptojanin, which is proline-rich, contains several SH3 domain consensus-binding sites and in fact, amphiphysin I and II interact in vitro with synaptojanin through distinct SH3 binding sequences w76x. Synaptojanin is highly enriched in brain and is concentrated in the presynaptic nerve terminal w100x where, like dynamin and amphiphysin, it is found in part on clathrin-coated endocytic structures w101x implicating a functional role for synaptojanin in endocytosis in the nerve terminal. As synaptojanin is a component of the amphiphysin IrII endocytic targeting complex, it appears likely that amphiphysin I and II function to target synaptojanin as well as dynamin to the endocytic site. In order to identify novel proteins potentially involved in regulating synaptojanin function, we used a biochemical screen to identify brain proteins that interact directly with synaptojanin. In addition to amphiphysin I and II, we identified a 40 kDa protein Žtermed endophilin. as the major synaptojanin-binding protein in the brain w102x. Partial purification of the protein, followed by peptide sequence analysis, revealed it to be identical to SH3P4, a protein that was identified as a member of a novel family of SH3 domain-containing proteins Žalong with SH3P8 and SH3P13. through a screen of an expression library with a Src class II SH3 domain peptide ligand w66x. SH3P4rendophilin was simultaneously identified as a major synaptojanin-binding protein through a yeast two-hybrid screen with the proline-rich C-terminus of synaptojanin w103x. Endophilin binds through its SH3 domain to a site in the proline-rich C-terminus of synaptojanin which is distinct from the sites for amphiphysin I and II w76x. Endophilin is brain specific and is highly concentrated in the presynaptic nerve terminal where it associates in a stable complex with synaptojanin in vivo w76, 103x. The functional role of the synaptojaninrendophilin complex is currently unknown. However, it may function to regulate the amount of synaptojanin at the endocytic site by regulating its accessibility for the amphiphysin IrII endocytic targeting complex. Alternatively, endophilin may not simply buffer excess synaptojanin, but may actively target it to other sites within the neuron through its central domain which has a high probability of participating in coiled-coil interactions.
Protein]Protein Interactions in Synaptic Vesicle Endocytosis
EH DOMAINS IN ENDOCYTOSIS: EVIDENCE FOR AN EH DOMAIN-SH3 DOMAIN CONNECTION Recently, attention has focused on a new protein]protein interaction domain that appears to play a role in endocytosis. This 60]70 amino acid module was first recognised in the protein Eps15 and has been referred to as the Eps15 homology ŽEH. domain w104x. Eps15, which contains three copies of the EH domain at its N-terminus, is constitutively bound through its C-terminus to the aadaptin subunit of AP2 w105]107x. Eps15-AP2 interactions are regulated by clathrin assembly w108x and Eps15 has been localised to the rim of clathrin-coated pits w109x. In Saccharomyces cerevisiae, End3p and Pan1p are required for endocytosis and contain one and two copies of the EH domain, respectively w110]113x. The EH domain appears to function as a protein]protein interaction module w104x and the amino acids asparagine-proline-phenylalanine ŽNPF. form the core of an EH domain-binding motif w114]117x. An interesting connection between EH domains and SH3 domains has now arisen from two independent observations. First, it has been demonstrated that synaptojanin-170, an alternatively spliced form of synaptojanin with a broad tissue distribution w90, 118x, binds to the EH domains of Eps15 in vitro w101x. Synaptojanin-170 has an identical domain structure as synaptojanin Žincluding the proline-rich sites that interact with SH3 domain-containing proteins. but contains an additional 25 kDa C-terminal region with three NPF repeats that mediate the EH domain interaction w101, 119x. It is interesting to note that the EH domain-containing yeast PAN1 gene interacts genetically with SJL1 w120x, one of three synaptojanin-like proteins in yeast w121x. A second link between EH domains and SH3 domains has come with the identification of intersectin, a protein composed of two N-terminal EH domains, a central helix forming region, and five C-terminal SH3 domains w117, 122x. The EH domains of intersectin also interact with the NPF core motif and interestingly, a subset of the SH3 domains interact with dynamin w117x. Intersectin is homologous with Dap160, a Drosophila protein with two EH domains and four SH3 domains that was identified based on its affinity for dynamin w123x. Screening of a mouse expression library with the intersectin EH domains has identified two novel intersectin-binding proteins ŽIbps. w117x. The Ibps are the mouse homolgues of rat epsin, a protein identified based on its interactions with the EH domains of Eps15 w124x. Epsin appears to be localised to clathrin-coated pits in situ, likely through its binding to the coat protein AP2 w124x. In addition to the presence of AP2-binding sites and multiple copies of the NPF motif, it is intriguing to note that the Ibpsrepsin contain the consensus clathrin-binding domain previously discussed. In fact, a protein complex containing intersectin with
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dynamin, AP2 and clathrin can be isolated from rat brain Žour unpublished results.. Thus, it appears that intersectin may function as an adaptor protein in endocytosis by bringing together novel clathrinrEH domain-binding proteins with SH3 domain-binding proteins such as dynamin.
CONCLUSIONS Both dynamin and synaptojanin, which have been localised to endocytic intermediates in the nerve terminal, appear to play critical roles in the formation of CCVs from the plasma membrane. As both are soluble proteins present primarily in the cytoplasm, it is important to understand the mechanisms that allow them to transiently localise to endocytic sites. In several different systems, SH3 domain-containing adaptor proteins function by localising their appropriate SH3 domain-binding partner to a specific subcellular localisation Žsuch as the plasma membrane. w22x. Recent molecular and biochemical data has identified several SH3 domain-containing proteins that appear to function as molecular adaptors in endocytosis. Thus, amphiphysin I and II, which interact through their SH3 domains with dynamin and synaptojanin, likely function by targeting these two key enzymes to endocytic domains through non-SH3 domain interactions with AP2 and clathrin. Endophilin may function by regulating this targeting complex or by mediating the targeting of synaptojanin and dynamin to distinct membrane or cytoskeletal sites. Intersectin may be involved in organising SH3 domain- and EH domain-binding proteins at the endocytic site. Research over the last several years has begun to reveal an unanticipated molecular complexity in clathrin-mediated endocytosis. This appears to be in contrast to some other vesicular budding events, such as the formation of COPII vesicles from the endoplasmic reticulum, which appears to require relatively few protein components w125x. Perhaps this complexity reflects the multiple roles for clathrin-mediated vesicular budding. Whereas COPII vesicles are used at a specific stage of vesicular trafficking w125x, clathrin-coated vesicles are used for budding from the TGN as well as for endocytosis from the plasma membrane. Further, in addition to synaptic vesicle membranes, a diverse array of proteins including receptors, ion channels, and membrane transporters undergo clathrinmediated endocytosis in both a regulated and constitutive manner. Complexity may also arise from the high demand for speed and efficiency placed on the endocytic machinery by SV endocytosis. Thus, neuronal cells express most endocytic proteins at much higher levels than non-neuronal cells and often express neuron specific isoforms w126x. To fully understand clathrin-mediated endocytosis and its role in the endocytosis of SVs, it will be critical to identify
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the full complement of proteins functioning in this process at which time we can begin to develop more precise molecular models which will eventually lead to a more complete understanding of this complex and fundamental event.
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