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Endophilin-1: a multifunctional protein
The International Journal of Biochemistry & Cell Biology 34 (2002) 1173–1177
Molecules in focus
Endophilin-1: a multifunctional protein Anne T. Reutens∗ , C. Glenn Begley Centre for Child Health Research and the Western Australian Institute for Medical Research, The University of WA, Telethon Institute for Child Health Research, 100 Roberts Road, Subiaco, WA 6008, Australia Received 22 January 2002; received in revised form 23 April 2002; accepted 23 April 2002
Abstract Endophilin-1, a cytoplasmic Src homology 3 (SH3) domain-containing protein, localises in brain presynaptic nerve termini. Endophilin dimerises through its N-terminus, and participates at multiple stages in clathrin-coated endocytosis, from early membrane invagination to synaptic vesicle uncoating. Both its C-terminal SH3 domain and N-terminus are required for endocytosis. Through its SH3 domain, endophilin bound to proline-rich domains (PRDs) in other endocytic proteins, including synaptojanin and dynamin. The N-terminal region possesses unique functions affecting lipid membrane curvature, through lysophosphatidic acid acyl transferase (LPAAT) activity and direct binding and tubulating activity. In addition to synaptic vesicle formation, endophilin-1 complexes with signalling molecules, including cell surface receptors, metalloprotease disintegrins and germinal centre kinase-like kinase (GLK). Therefore, endophilin-1 may serve to couple vesicle biogenesis with intracellular signalling cascades. © 2002 Elsevier Science Ltd. All rights reserved. Keywords: Endophilin-1; Endocytosis; Tyrosine kinase; SH3 domain
1. Introduction
2. Structure
Endophilin-1 was first identified because of it Src homology 3 (SH3) domain [1,2] and via its expression in brain and interaction with synaptojanin [3]. Endophilin-1 was also known as SH3p4, SH3GL2, CNSA2, EEN-B1 and SH3D2A.
The human endophilin-1 gene mapped to 9p22 [2]. The gene of nine exons encoded a 352 amino acid (aa) 40 kDa protein (GenBank GI: 1869812). Alignment with two other closely related proteins, endophilin-2 and endophilin-3, (SH3p8/SH3GL1 and SH3p13/SH3GL3, respectively) identified a unique SH3 family. The three proteins share a 260 aa conserved N-terminal domain with predicted ␣ helical and coiled-coil conformation, the C-terminal SH3 domain (aa 295–344) 70% homologous with the adaptor protein GRB2, and a short hinge region [2,3] (Fig. 1). Endophilin-1 existed as an 80–90 kDa homodimer in rat brain cytosol [4]. Dimerisation required the coiled-coil region (aa 125–290). Endophilin-1 also heterodimerised with endophilin-2.
Abbreviations: aa, amino acid; GLK, germinal centre kinaselike kinase; JNK, c-Jun N-terminal kinase; LPAAT, lysophosphatidic acid acyl transferase; PRD, proline-rich domain; SH3, Src homology 3; SLMV, synaptic-like microvesicle; CIN85, Cbl-interacting protein of 85 kDa ∗ Corresponding author. Tel.: +61-8-9489-7888; fax: +61-8-9489-7700. E-mail address: [email protected] (A.T. Reutens).
1357-2725/02/$ – see front matter © 2002 Elsevier Science Ltd. All rights reserved. PII: S 1 3 5 7 - 2 7 2 5 ( 0 2 ) 0 0 0 6 3 - 8
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A.T. Reutens, C.G. Begley / The International Journal of Biochemistry & Cell Biology 34 (2002) 1173–1177
Fig. 1. Schematic representation of the domain structure of endophilin-1. The SH3 domain is indicated in black, the hinge region in stripes and the N-terminal ␣ helical and coiled-coil domain in grey. The locations of the dimerisation domain, lysophosphatidic acid acyl transferase active region and putative nuclear localisation domain are shown beneath the N-terminal domain. The numbers refer to the amino acid residue positions of the various domains.
3. Synthesis and degradation Factors determining endophilin-1 expression or degradation have not been reported. The human endophilin-1 gene promoter was TATA-less, with multiple potential transcription factor binding sites (e.g. Sp1, NF-kappaB, ATF, c-Jun). Endophilin-1 was predominantly expressed in the brain [2,3], particularly in foetal cerebellum at 20 weeks gestation and in adult frontal cortex [2]. Although aa 159–174 contained a nuclear localisation motif, endophilin-1 showed a punctate and diffuse cytosolic staining pattern, enriched in presynaptic neurons [5]. Endophilin-1 colocalised in nerve terminal cytoplasm with proteins including dynamin, a 100 kDa GTPase; synaptojanin, a presynaptic 145 kDa inositol 5-phosphatase; and amphiphysin, a SH3-domain containing protein [3].
4. Biological function 4.1. Endocytic functions of endophilin-1 Endophilin-1 affects the clathrin-coated vesicle endocytic pathway regulating synaptic vesicle formation (reviewed in [6]). It participates in invagination, fission and vesicle recycling. Its critical role has been
demonstrated in several models: (1) Depletion of cytosolic endophilin-1 decreased synaptic-like microvesicle (SLMV) biogenesis from deep plasma membrane invaginations [7]. (2) A glutathione S-transferase fusion protein containing only the SH3 domain of endophilin-1 inhibited endocytosis [8] and interfered with invagination and vesicle scission [9]. (3) Anti-endophilin-1 antibody injection into presynaptic compartments of stimulated lamprey synapses depleted synaptic vesicles and caused shallow coated pit accumulation [10,11]. (4) Endophilin-depleted brain cytosol had fewer dynamin-coated structures [10]. (5) Peptide blockade of endophilin SH3 domain function disrupted vesicle recycling; accumulation of deeply invaginated pits and free clathrin-coated vesicles suggested a role in vesicle uncoating [11]. Thus, endophilin-1 is implicated in both early and late stages of endocytosis. Endophilin-1’s actions probably reflect the functions of its SH3 and N-terminal domains. For example, the SH3 domain is critical for interaction with synaptojanin-1; is required for endocytosis [7]; is assembled with dynamin in rings around necks of clathrin-coated pits; is involved in membrane scission and vesicle release [3]. In contrast, the N-terminal portion of endophilin-1 was recently shown to have lysophosphatidic acid acyl transferase (LPAAT) activity [7] by which arachi-
A.T. Reutens, C.G. Begley / The International Journal of Biochemistry & Cell Biology 34 (2002) 1173–1177
donate transfer to lysophosphatidic acid generated phosphatidic acid. Endophilin-1 preferentially utilised longer chain unsaturated fatty acyl CoA as substrate for this reaction. Conversion of the inverted coneshaped lysophosphatidic acid into cone-shaped phosphatidic acid was hypothesised to induce positive-tonegative lipid membrane curvature required for vesicle formation. However, in addition to LPAAT activity, the SH3 domain of endophilin was still critical for SLMV biogenesis, indicating the importance of endophilin-1 protein–protein interactions [7]. However, other workers argued that LPAAT activity was dispensable for membrane deformation [12]. Endophilin-1 induced tubule formation in synthetic
1175
liposomes lacking its putative LPAAT substrates, and when the putative enzymatic activity was inhibited. Tubulation was induced by a 29 aa stretch in the N-terminal first 35 residues, possessing an amphipathic pattern with a hydrophobic patch and hydrophilic face [12]. This region and the SH3 domain were required to form endophilin-1/dynamin ring complexes along tubules, with endophilin-1 inhibiting dynamin’s GTP-dependent vesiculating activity. In this model, endophilin-1 was targeted to lipid bilayers, tubulated the membrane and oligomerised with other membrane proteins. However, this model does not necessarily exclude the lipid-modifying model. Either or both models for endophilin N-terminal function may apply.
Fig. 2. Models for endophilin-1 activity. Endophilin-1 affects the clathrin-coated vesicle endocytic pathway, at steps from invagination through to vesicle uncoating. Shown inset are the properties of the N-terminal domain of endophilin-1 required for endocytosis: binding to lipid bilayers, tubulation and lysophosphatidic acid acyl transferase activity (LPAAT), which converts lysophosphatidic acid (LPA) to phosphatidic acid (PA). The SH3 domain interacts with other endocytic proteins such as dynamin and synaptojanin, and modulates signalling pathways by protein–protein interactions.
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A.T. Reutens, C.G. Begley / The International Journal of Biochemistry & Cell Biology 34 (2002) 1173–1177
4.2. Non-endocytic functions of endophilin-1 Endophilin-1 interacts with numerous non-endocytic proteins. The SH3 domain of endophilin-1 bound to a 24 aa proline-rich domain (PRD) in the third intracellular loop of the G-protein-coupled 1-adrenergic receptor [13]. Endophilin-1 overexpression increased isoproterenol-induced receptor internalisation by 25% and decreased coupling of receptor to the G-protein. Endophilin’s SH3 domain also bound to a PRD within the cytoplasmic tail of metalloprotease disintegrins, transmembrane glycoproteins acting in cell adhesion and growth factor signalling [14]. Endophilin-1 bound preferentially to the pro-form found in the trans-Golgi network. Therefore, endophilin-1 binding may regulate metalloprotease disintegrin intracellular transit and maturation. Rat germinal centre kinase-like kinase (rGLK), a serine/ threonine cytosolic kinase, interacted with endophilin-1 [15]. rGLK modulated c-Jun N-terminal kinase (JNK) activity by phosphorylation and bound the SH3 domain of endophilin-1 through a C-terminal PRD. Coexpression of rGLK and full-length endophilin-1 increased JNK activity two-fold, whereas coexpression with the endophilin SH3 domain abrogated rGLK-induced JNK activation. Endophilin-1, therefore, modulated the mitogen-activated protein kinase pathway through physical association with rGLK. Thus, endophilin-1 may help to recruit and localise intracellular signalling components. The SH3 domain of endophilin-1 also bound Cbl-interacting protein of 85 kDa (CIN85). This complex, when recruited by Cbl, influences internalisation, degradation and intracellular signalling of tyrosine kinase receptors for hepatocyte [16] and epidermal growth factors [17]. Unlike endophilin-1, endophilin-2 is found in a multitude of tissues, and endophilin-3 is localised to brain and testis. Endophilin-2 and endophilin-3 bound to synaptojanin, dynamin [3] and the 1-adrenergic receptor [13]. However, evidence of membrane lipid bilayer deforming ability or LPAAT activity for endophilin-2 and endophilin-3 has not been reported. In summary, endophilin-1 was essential for synaptic vesicle endocytosis and regulated intracellular signalling pathways, perhaps by controlling activation and compartmentalisation (Fig. 2). This fascinating
multifunctional protein serves as a good example of proteins that have different functions depending on the partners with which they interact, and their location within a cell.
5. Possible medical applications No human disease associated with abnormal endophilin-1 function has been reported. Since endophilin-1 interacted with multiple proteins and affected synaptic endocytosis, such a disease could potentially result from blockade of synaptic function. References [1] A.B. Sparks, N.G. Hoffman, S.J. McConnell, D.M. Fowlkes, B.K. Kay, Cloning of ligand targets: systematic isolation of SH3 domain-containing proteins, Nat. Biotechnol. 14 (1996) 741–744. [2] C. Giachino, E. Lantelme, L. Lanzetti, S. Saccone, G. Della Valle, N. Migone, A novel SH3-containing human gene family preferentially expressed in the central nervous system, Genomics 41 (1997) 427–434. [3] N. Ringstad, Y. Nemoto, P. De Camilli, The SH3p4/SH3p8/ SH3p13 protein family: binding partners for synaptojanin and dynamin via a Grb2-like Src homology 3 domain, Proc. Natl. Acad. Sci. U.S.A. 94 (1997) 8569–8574. [4] N. Ringstad, Y. Nemoto, P. De Camilli, Differential expression of endophilin-1 and endophilin-2 dimers at central nervous system synapses, J. Biol. Chem. 276 (2001) 40424–40430. [5] K.D. Micheva, B.K. Kay, P.S. McPherson, Synaptojanin forms two separate complexes in the nerve terminal: interactions with endophilin and amphiphysin, J. Biol. Chem. 272 (1997) 27239–27245. [6] M.J. Hannah, A.A. Schmidt, W.B. Huttner, Synaptic vesicle biogenesis, Annu. Rev. Cell Dev. Biol. 15 (1999) 733–798. [7] A. Schmidt, M. Wolde, C. Thiele, W. Fest, H. Kratzin, A.V. Podtelejnikov, W. Witke, W.B. Huttner, H.D. Söling, Endophilin-1 mediates synaptic vesicle formation by transfer of arachidonate to lysophosphatidic acid, Nature 401 (1999) 133–141. [8] F. Simpson, N.K. Hussain, B. Qualmann, R.B. Kelly, B.K. Kay, P.S. McPherson, S.L. Schmid, SH3-domain-containing proteins function at distinct steps in clathrin-coated vesicle formation, Nat. Cell Biol. 1 (1999) 119–124. [9] E. Hill, J. Van der Kaay, C.P. Downes, E. Smythe, The role of dynamin and its binding partners in coated pit invagination and scission, J. Cell Biol. 152 (2001) 309–323. [10] N. Ringstad, H. Gad, P. Löw, G. Di Paolo, L. Brodin, O. Shupliakov, P. De Camilli, Endophilin/SH3p4 is required for
A.T. Reutens, C.G. Begley / The International Journal of Biochemistry & Cell Biology 34 (2002) 1173–1177 the transition from early to late stages in clathrin-mediated synaptic vesicle endocytosis, Neuron 24 (1999) 143–154. [11] H. Gad, N. Ringstad, P. Löw, O. Kjaerulff, J. Gustafson, M. Wenk, G. Di Paolo, Y. Nemoto, J. Crum, M.H. Ellisman, P. De Camilli, O. Shupliakov, L. Brodin, Fission and uncoating of synaptic clathrin-coated vesicle are perturbed by disruption of interactions with the SH3 domain of endophilin, Neuron 27 (2000) 301–312. [12] K. Farsad, N. Ringstad, K. Takei, S.R. Floyd, K. Rose, P. De Camilli, Generation of high curvature membranes mediated by direct endophilin bilayer interactions, J. Cell Biol. 155 (2001) 193–200. [13] Y. Tang, L.A. Hu, W.E. Miller, N. Ringstad, R.A. Hall, J.A. Pitcher, P. DeCamilli, R.J. Lefkowitz, Identification of the endophilins (SH3p4/p8/p13) as novel binding partners for the 1-adrenergic receptor, Proc. Natl. Acad. Sci. U.S.A. 96 (1999) 12559–12564.
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[14] L. Howard, K.K. Nelson, R.A. Maciewicz, C.P. Blobel, Interaction of the metalloprotease disintegrins MDC9 and MDC15 with two SH3 domain-containing proteins, endophilin-1 and SH3PX1, J. Biol. Chem. 274 (1999) 31693– 31699. [15] A.R. Ramjaun, A. Angers, V. Legendre-Guillemin, X.-K. Tong, P.S. McPherson, Endophilin regulates JNK activation through its interaction with the germinal center kinase-like kinase, J. Biol. Chem. 276 (2001) 28913–28919. [16] A. Petrelli, G.F. Gilestro, S. Lanzardo, P.M. Comoglio, N. Migone, S. Giordano, The endophilin–CIN85–Cbl complex mediates ligand-dependent downregulation of c-Met, Nature 416 (2002) 187–190. [17] P. Soubeyran, K. Kowanetz, I. Szymkiewicz, W.Y. Langdon, I. Dikic, Cbl–CIN85–endophilin complex mediates ligandinduced downregulation of EGF receptors, Nature 416 (2002) 183–187.
Molecules in focus
Endophilin-1: a multifunctional protein Anne T. Reutens∗ , C. Glenn Begley Centre for Child Health Research and the Western Australian Institute for Medical Research, The University of WA, Telethon Institute for Child Health Research, 100 Roberts Road, Subiaco, WA 6008, Australia Received 22 January 2002; received in revised form 23 April 2002; accepted 23 April 2002
Abstract Endophilin-1, a cytoplasmic Src homology 3 (SH3) domain-containing protein, localises in brain presynaptic nerve termini. Endophilin dimerises through its N-terminus, and participates at multiple stages in clathrin-coated endocytosis, from early membrane invagination to synaptic vesicle uncoating. Both its C-terminal SH3 domain and N-terminus are required for endocytosis. Through its SH3 domain, endophilin bound to proline-rich domains (PRDs) in other endocytic proteins, including synaptojanin and dynamin. The N-terminal region possesses unique functions affecting lipid membrane curvature, through lysophosphatidic acid acyl transferase (LPAAT) activity and direct binding and tubulating activity. In addition to synaptic vesicle formation, endophilin-1 complexes with signalling molecules, including cell surface receptors, metalloprotease disintegrins and germinal centre kinase-like kinase (GLK). Therefore, endophilin-1 may serve to couple vesicle biogenesis with intracellular signalling cascades. © 2002 Elsevier Science Ltd. All rights reserved. Keywords: Endophilin-1; Endocytosis; Tyrosine kinase; SH3 domain
1. Introduction
2. Structure
Endophilin-1 was first identified because of it Src homology 3 (SH3) domain [1,2] and via its expression in brain and interaction with synaptojanin [3]. Endophilin-1 was also known as SH3p4, SH3GL2, CNSA2, EEN-B1 and SH3D2A.
The human endophilin-1 gene mapped to 9p22 [2]. The gene of nine exons encoded a 352 amino acid (aa) 40 kDa protein (GenBank GI: 1869812). Alignment with two other closely related proteins, endophilin-2 and endophilin-3, (SH3p8/SH3GL1 and SH3p13/SH3GL3, respectively) identified a unique SH3 family. The three proteins share a 260 aa conserved N-terminal domain with predicted ␣ helical and coiled-coil conformation, the C-terminal SH3 domain (aa 295–344) 70% homologous with the adaptor protein GRB2, and a short hinge region [2,3] (Fig. 1). Endophilin-1 existed as an 80–90 kDa homodimer in rat brain cytosol [4]. Dimerisation required the coiled-coil region (aa 125–290). Endophilin-1 also heterodimerised with endophilin-2.
Abbreviations: aa, amino acid; GLK, germinal centre kinaselike kinase; JNK, c-Jun N-terminal kinase; LPAAT, lysophosphatidic acid acyl transferase; PRD, proline-rich domain; SH3, Src homology 3; SLMV, synaptic-like microvesicle; CIN85, Cbl-interacting protein of 85 kDa ∗ Corresponding author. Tel.: +61-8-9489-7888; fax: +61-8-9489-7700. E-mail address: [email protected] (A.T. Reutens).
1357-2725/02/$ – see front matter © 2002 Elsevier Science Ltd. All rights reserved. PII: S 1 3 5 7 - 2 7 2 5 ( 0 2 ) 0 0 0 6 3 - 8
1174
A.T. Reutens, C.G. Begley / The International Journal of Biochemistry & Cell Biology 34 (2002) 1173–1177
Fig. 1. Schematic representation of the domain structure of endophilin-1. The SH3 domain is indicated in black, the hinge region in stripes and the N-terminal ␣ helical and coiled-coil domain in grey. The locations of the dimerisation domain, lysophosphatidic acid acyl transferase active region and putative nuclear localisation domain are shown beneath the N-terminal domain. The numbers refer to the amino acid residue positions of the various domains.
3. Synthesis and degradation Factors determining endophilin-1 expression or degradation have not been reported. The human endophilin-1 gene promoter was TATA-less, with multiple potential transcription factor binding sites (e.g. Sp1, NF-kappaB, ATF, c-Jun). Endophilin-1 was predominantly expressed in the brain [2,3], particularly in foetal cerebellum at 20 weeks gestation and in adult frontal cortex [2]. Although aa 159–174 contained a nuclear localisation motif, endophilin-1 showed a punctate and diffuse cytosolic staining pattern, enriched in presynaptic neurons [5]. Endophilin-1 colocalised in nerve terminal cytoplasm with proteins including dynamin, a 100 kDa GTPase; synaptojanin, a presynaptic 145 kDa inositol 5-phosphatase; and amphiphysin, a SH3-domain containing protein [3].
4. Biological function 4.1. Endocytic functions of endophilin-1 Endophilin-1 affects the clathrin-coated vesicle endocytic pathway regulating synaptic vesicle formation (reviewed in [6]). It participates in invagination, fission and vesicle recycling. Its critical role has been
demonstrated in several models: (1) Depletion of cytosolic endophilin-1 decreased synaptic-like microvesicle (SLMV) biogenesis from deep plasma membrane invaginations [7]. (2) A glutathione S-transferase fusion protein containing only the SH3 domain of endophilin-1 inhibited endocytosis [8] and interfered with invagination and vesicle scission [9]. (3) Anti-endophilin-1 antibody injection into presynaptic compartments of stimulated lamprey synapses depleted synaptic vesicles and caused shallow coated pit accumulation [10,11]. (4) Endophilin-depleted brain cytosol had fewer dynamin-coated structures [10]. (5) Peptide blockade of endophilin SH3 domain function disrupted vesicle recycling; accumulation of deeply invaginated pits and free clathrin-coated vesicles suggested a role in vesicle uncoating [11]. Thus, endophilin-1 is implicated in both early and late stages of endocytosis. Endophilin-1’s actions probably reflect the functions of its SH3 and N-terminal domains. For example, the SH3 domain is critical for interaction with synaptojanin-1; is required for endocytosis [7]; is assembled with dynamin in rings around necks of clathrin-coated pits; is involved in membrane scission and vesicle release [3]. In contrast, the N-terminal portion of endophilin-1 was recently shown to have lysophosphatidic acid acyl transferase (LPAAT) activity [7] by which arachi-
A.T. Reutens, C.G. Begley / The International Journal of Biochemistry & Cell Biology 34 (2002) 1173–1177
donate transfer to lysophosphatidic acid generated phosphatidic acid. Endophilin-1 preferentially utilised longer chain unsaturated fatty acyl CoA as substrate for this reaction. Conversion of the inverted coneshaped lysophosphatidic acid into cone-shaped phosphatidic acid was hypothesised to induce positive-tonegative lipid membrane curvature required for vesicle formation. However, in addition to LPAAT activity, the SH3 domain of endophilin was still critical for SLMV biogenesis, indicating the importance of endophilin-1 protein–protein interactions [7]. However, other workers argued that LPAAT activity was dispensable for membrane deformation [12]. Endophilin-1 induced tubule formation in synthetic
1175
liposomes lacking its putative LPAAT substrates, and when the putative enzymatic activity was inhibited. Tubulation was induced by a 29 aa stretch in the N-terminal first 35 residues, possessing an amphipathic pattern with a hydrophobic patch and hydrophilic face [12]. This region and the SH3 domain were required to form endophilin-1/dynamin ring complexes along tubules, with endophilin-1 inhibiting dynamin’s GTP-dependent vesiculating activity. In this model, endophilin-1 was targeted to lipid bilayers, tubulated the membrane and oligomerised with other membrane proteins. However, this model does not necessarily exclude the lipid-modifying model. Either or both models for endophilin N-terminal function may apply.
Fig. 2. Models for endophilin-1 activity. Endophilin-1 affects the clathrin-coated vesicle endocytic pathway, at steps from invagination through to vesicle uncoating. Shown inset are the properties of the N-terminal domain of endophilin-1 required for endocytosis: binding to lipid bilayers, tubulation and lysophosphatidic acid acyl transferase activity (LPAAT), which converts lysophosphatidic acid (LPA) to phosphatidic acid (PA). The SH3 domain interacts with other endocytic proteins such as dynamin and synaptojanin, and modulates signalling pathways by protein–protein interactions.
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A.T. Reutens, C.G. Begley / The International Journal of Biochemistry & Cell Biology 34 (2002) 1173–1177
4.2. Non-endocytic functions of endophilin-1 Endophilin-1 interacts with numerous non-endocytic proteins. The SH3 domain of endophilin-1 bound to a 24 aa proline-rich domain (PRD) in the third intracellular loop of the G-protein-coupled 1-adrenergic receptor [13]. Endophilin-1 overexpression increased isoproterenol-induced receptor internalisation by 25% and decreased coupling of receptor to the G-protein. Endophilin’s SH3 domain also bound to a PRD within the cytoplasmic tail of metalloprotease disintegrins, transmembrane glycoproteins acting in cell adhesion and growth factor signalling [14]. Endophilin-1 bound preferentially to the pro-form found in the trans-Golgi network. Therefore, endophilin-1 binding may regulate metalloprotease disintegrin intracellular transit and maturation. Rat germinal centre kinase-like kinase (rGLK), a serine/ threonine cytosolic kinase, interacted with endophilin-1 [15]. rGLK modulated c-Jun N-terminal kinase (JNK) activity by phosphorylation and bound the SH3 domain of endophilin-1 through a C-terminal PRD. Coexpression of rGLK and full-length endophilin-1 increased JNK activity two-fold, whereas coexpression with the endophilin SH3 domain abrogated rGLK-induced JNK activation. Endophilin-1, therefore, modulated the mitogen-activated protein kinase pathway through physical association with rGLK. Thus, endophilin-1 may help to recruit and localise intracellular signalling components. The SH3 domain of endophilin-1 also bound Cbl-interacting protein of 85 kDa (CIN85). This complex, when recruited by Cbl, influences internalisation, degradation and intracellular signalling of tyrosine kinase receptors for hepatocyte [16] and epidermal growth factors [17]. Unlike endophilin-1, endophilin-2 is found in a multitude of tissues, and endophilin-3 is localised to brain and testis. Endophilin-2 and endophilin-3 bound to synaptojanin, dynamin [3] and the 1-adrenergic receptor [13]. However, evidence of membrane lipid bilayer deforming ability or LPAAT activity for endophilin-2 and endophilin-3 has not been reported. In summary, endophilin-1 was essential for synaptic vesicle endocytosis and regulated intracellular signalling pathways, perhaps by controlling activation and compartmentalisation (Fig. 2). This fascinating
multifunctional protein serves as a good example of proteins that have different functions depending on the partners with which they interact, and their location within a cell.
5. Possible medical applications No human disease associated with abnormal endophilin-1 function has been reported. Since endophilin-1 interacted with multiple proteins and affected synaptic endocytosis, such a disease could potentially result from blockade of synaptic function. References [1] A.B. Sparks, N.G. Hoffman, S.J. McConnell, D.M. Fowlkes, B.K. Kay, Cloning of ligand targets: systematic isolation of SH3 domain-containing proteins, Nat. Biotechnol. 14 (1996) 741–744. [2] C. Giachino, E. Lantelme, L. Lanzetti, S. Saccone, G. Della Valle, N. Migone, A novel SH3-containing human gene family preferentially expressed in the central nervous system, Genomics 41 (1997) 427–434. [3] N. Ringstad, Y. Nemoto, P. De Camilli, The SH3p4/SH3p8/ SH3p13 protein family: binding partners for synaptojanin and dynamin via a Grb2-like Src homology 3 domain, Proc. Natl. Acad. Sci. U.S.A. 94 (1997) 8569–8574. [4] N. Ringstad, Y. Nemoto, P. De Camilli, Differential expression of endophilin-1 and endophilin-2 dimers at central nervous system synapses, J. Biol. Chem. 276 (2001) 40424–40430. [5] K.D. Micheva, B.K. Kay, P.S. McPherson, Synaptojanin forms two separate complexes in the nerve terminal: interactions with endophilin and amphiphysin, J. Biol. Chem. 272 (1997) 27239–27245. [6] M.J. Hannah, A.A. Schmidt, W.B. Huttner, Synaptic vesicle biogenesis, Annu. Rev. Cell Dev. Biol. 15 (1999) 733–798. [7] A. Schmidt, M. Wolde, C. Thiele, W. Fest, H. Kratzin, A.V. Podtelejnikov, W. Witke, W.B. Huttner, H.D. Söling, Endophilin-1 mediates synaptic vesicle formation by transfer of arachidonate to lysophosphatidic acid, Nature 401 (1999) 133–141. [8] F. Simpson, N.K. Hussain, B. Qualmann, R.B. Kelly, B.K. Kay, P.S. McPherson, S.L. Schmid, SH3-domain-containing proteins function at distinct steps in clathrin-coated vesicle formation, Nat. Cell Biol. 1 (1999) 119–124. [9] E. Hill, J. Van der Kaay, C.P. Downes, E. Smythe, The role of dynamin and its binding partners in coated pit invagination and scission, J. Cell Biol. 152 (2001) 309–323. [10] N. Ringstad, H. Gad, P. Löw, G. Di Paolo, L. Brodin, O. Shupliakov, P. De Camilli, Endophilin/SH3p4 is required for
A.T. Reutens, C.G. Begley / The International Journal of Biochemistry & Cell Biology 34 (2002) 1173–1177 the transition from early to late stages in clathrin-mediated synaptic vesicle endocytosis, Neuron 24 (1999) 143–154. [11] H. Gad, N. Ringstad, P. Löw, O. Kjaerulff, J. Gustafson, M. Wenk, G. Di Paolo, Y. Nemoto, J. Crum, M.H. Ellisman, P. De Camilli, O. Shupliakov, L. Brodin, Fission and uncoating of synaptic clathrin-coated vesicle are perturbed by disruption of interactions with the SH3 domain of endophilin, Neuron 27 (2000) 301–312. [12] K. Farsad, N. Ringstad, K. Takei, S.R. Floyd, K. Rose, P. De Camilli, Generation of high curvature membranes mediated by direct endophilin bilayer interactions, J. Cell Biol. 155 (2001) 193–200. [13] Y. Tang, L.A. Hu, W.E. Miller, N. Ringstad, R.A. Hall, J.A. Pitcher, P. DeCamilli, R.J. Lefkowitz, Identification of the endophilins (SH3p4/p8/p13) as novel binding partners for the 1-adrenergic receptor, Proc. Natl. Acad. Sci. U.S.A. 96 (1999) 12559–12564.
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[14] L. Howard, K.K. Nelson, R.A. Maciewicz, C.P. Blobel, Interaction of the metalloprotease disintegrins MDC9 and MDC15 with two SH3 domain-containing proteins, endophilin-1 and SH3PX1, J. Biol. Chem. 274 (1999) 31693– 31699. [15] A.R. Ramjaun, A. Angers, V. Legendre-Guillemin, X.-K. Tong, P.S. McPherson, Endophilin regulates JNK activation through its interaction with the germinal center kinase-like kinase, J. Biol. Chem. 276 (2001) 28913–28919. [16] A. Petrelli, G.F. Gilestro, S. Lanzardo, P.M. Comoglio, N. Migone, S. Giordano, The endophilin–CIN85–Cbl complex mediates ligand-dependent downregulation of c-Met, Nature 416 (2002) 187–190. [17] P. Soubeyran, K. Kowanetz, I. Szymkiewicz, W.Y. Langdon, I. Dikic, Cbl–CIN85–endophilin complex mediates ligandinduced downregulation of EGF receptors, Nature 416 (2002) 183–187.