SH3 Domain Of Amphiphysin Research Articles

Identification and characterization of a novel mammalian isoform of the endocytic adaptor ITSN1

Intersectin 1 (ITSN1) is an evolutionarily conserved adaptor protein engaged in clathrin-mediated endocytosis, cell signaling and actin cytoskeleton rearrangements. Two major ITSN1 isoforms were initially described, the ubiquitous short isoform (ITSN1-s) and the long isoform (ITSN1-l) expressed predominantly in neurons. Numerous alternative splicing events for ITSN1 pre-mRNA were later identified. Here we describe a novel isoform ITSN1-22a with an alternative C-terminus encoded by exon 22a. This exon is only found in placental mammals. The transcript of ITSN1-22a is detected in a wide range of human and mouse tissues. We show here that two alternative splicing events affect the coding sequence of the ITSN1-22a isoform. Moreover, alternative polyadenylation of these transcripts was demonstrated in human tissues. The protein encoded by the ITSN1-22a transcript possesses two EH domains, a coiled-coil region, an SH3A domain and a specific C-terminal domain (CTD) but lacks four SH3 domains in comparison with ITSN1-s. The level of ITSN1-22a protein varies in different mouse tissues and human cell lines. The highest amounts of this isoform occur in mouse brain, spleen, lung and the human B cell line DG75. ITSN1-22a binds via its CTD to the SH3 domain of the endocytic protein amphiphysin 1 and the SH3A domain of ITSN1. Furthermore association in vivo and codistribution of ITSN1-22a and ITSN1-s were demonstrated suggesting that these isoforms could function in concert. We have revealed differential binding of ITSN1-s and ITSN1-22a to the ubiquitin ligase Cbl. Both isoforms possess the SH3A domain capable of binding to Cbl; however ITSN1-22a in contrast to ITSN1-s did not interact with Cbl in vivo. In vitro binding experiments demonstrated that the CTD of ITSN1-22a negatively regulated its binding to Cbl; at the same time interaction with another partner, dynamin 1 was not affected by the presence of the CTD. These data suggest that intramolecular interaction within ITSN1-22a could specifically regulate its binding to protein partners. Thus, this novel mammalian ITSN1 isoform possesses a significantly altered domain structure and performs specific protein–protein interactions.

Bioinformatics analysis of the structure and function correlated to interferon sensitivity of HCV core protein

To analyze and predict the structure and function correlated to interferon sensitivity of HCV Core protein in different genotypes. Using different bioinformafics software respectively to comparatively analyze and predict the secondary structure, tertiary structure, modification site and main functional motifs of HCV Core protein. There were diversities in secondary structure and tertiary structure of different genotypes. HCV Core protein would exist some modification sites about amidation, cAMP, PKC, TYR, and interact with amphiphysin SH3 domain, ERK, PKC. Differences in structure and function of HCV Core protein of different genotypes affected interferon sensitivity.

Applying Chemometrics Approaches to Model and Predict the Binding Affinities Between the Human Amphiphysin SH3 Domain and Its Peptide Ligands (Suplementary Material)

In this study, we used two categories of molecular descriptors as CODESSA and DPPS (divided physicochemical property scores of amino acids) to parameterize structural characteristics of 2015 human amphiphysin SH3 domain-binding decapeptides at atom and residue levels. Based upon that, several robust quantitative structure-affinity relationship (QSAR) models were then constructed using partial least squares regression (PLS) and least squares-support vector machine (LSSVM) coupled with genetic algorithm (GA)-variable selection. Results show that (1) GA is a powerful tool for variable selection by which the most informative variable combinations can be efficiently determined for PLS and LSSVM modeling, (2) regression models constructed using nonlinear LSSVM approach are more robust and predictable than those by linear PLS method, (3) the residue level descriptor (DPPS) performs better in capturing peptide structural characteristics, more amenable than those from the atom level descriptor (CODESSA). By investigating the optimal DPPS-based GA-LSSVM model, it is indicated that the core motif of SH3 domain-binding peptides contributes significantly to the binding affinity, whereas the two end residues, especially the N-terminal residue, have a little effect on the binding process.

Dynamic Interaction of Amphiphysin with N-WASP Regulates Actin Assembly

Amphiphysin 1, an endocytic adaptor concentrated at synapses that couples clathrin-mediated endocytosis to dynamin-dependent fission, was also shown to have a regulatory role in actin dynamics. Here, we report that amphiphysin 1 interacts with N-WASP and stimulates N-WASP- and Arp2/3-dependent actin polymerization. Both the Src homology 3 and the N-BAR domains are required for this stimulation. Acidic liposome-triggered, N-WASP-dependent actin polymerization is strongly impaired in brain cytosol of amphiphysin 1 knock-out mice. FRET-FLIM analysis of Sertoli cells, where endogenously expressed amphiphysin 1 co-localizes with N-WASP in peripheral ruffles, confirmed the association between the two proteins in vivo. This association undergoes regulation and is enhanced by stimulating phosphatidylserine receptors on the cell surface with phosphatidylserine-containing liposomes that trigger ruffle formation. These results indicate that actin regulation is a key function of amphiphysin 1 and that such function cooperates with the endocytic adaptor role and membrane shaping/curvature sensing properties of the protein during the endocytic reaction.

Antibodies against the amphiphysin sh-3 domain mediate reduced gabaergic motor inhibition and anxiety of stiff person syndrome

Antibodies against the amphiphysin sh-3 domain mediate reduced gabaergic motor inhibition and anxiety of stiff person syndrome

Modeling and prediction of binding affinities between the human amphiphysin SH3 domain and its peptide ligands using genetic algorithm‐Gaussian processes

In this article, we discuss the application of the Gaussian process (GP) and other statistical methods (PLS, ANN, and SVM) for the modeling and prediction of binding affinities between the human amphiphysin SH3 domain and its peptide ligands. Divided physicochemical property scores of amino acids, involving significant hydrogen bond, electronic, hydrophobic, and steric properties, was used to characterize the peptide structures, and quantitative structure-affinity relationship models were then constructed by PLS, ANN, SVM, and GP coupled with genetic algorithm-variable selection. The results show that: (i) since the significant flexibility and high complexity possessed in polypeptide structures, linear PLS method was incapable of fulfilling a satisfying behavior on SH3 domain binding peptide dataset; (ii) the overfitting involved in training process has decreased the predictive power of ANN model to some extent; (iii) both SVM and GP have a good performance for SH3 domain binding peptide dataset. Moreover, by combining linear and nonlinear terms in the covariance function, the GP is capable of handling linear and nonlinear-hybrid relationship, and which thus obtained a more stable and predictable model than SVM. Analyses of GP models showed that diversified properties contribute remarkable effect to the interactions between the SH3 domain and the peptides. Particularly, steric property and hydrophobicity of P(2), electronic property of P(0), and electronic property and hydrogen bond property of P(-3) in decapeptide (P(4)P(3)P(2)P(1)P(0)P(-1)P(-2)P(-3)P(-4)P(-5)) significantly contribute to the binding affinities of SH3 domain-peptide interactions.

Phosphorylation of Amphiphysin I by Minibrain Kinase/Dual-specificity Tyrosine Phosphorylation-regulated Kinase, a Kinase Implicated in Down Syndrome

Minibrain kinase/dual-specificity tyrosine phosphorylation-regulated kinase (Mnb/Dyrk1A) is a proline-directed serine/threonine kinase encoded in the Down syndrome critical region of human chromosome 21. This kinase has been shown to phosphorylate dynamin 1 and synaptojanin 1. Here we report that amphiphysin I (Amph I) is also a Mnb/Dyrk1A substrate. This kinase phosphorylated native Amph I in rodent brains and recombinant human Amph I expressed in Escherichia coli. Serine 293 (Ser-293) was identified as the major site, whereas serine 295 and threonine 310 were found as minor kinase sites. In cultured cells, recombinant Amph I was phosphorylated at Ser-293 by endogenous kinase(s). Because mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) has been suggested to phosphorylate Amph I at Ser-293, our efforts addressed whether Ser-293 is phosphorylated in vivo by MAPK/ERK or by Mnb/Dyrk1A. Overnight serum-withdrawal inactivated MAPK/ERK; nonetheless, Ser-293 was phosphorylated in Chinese hamster ovary and SY5Y cells. Epigallocatechin-3-gallate, a potent Mnb/Dyrk1A inhibitor in vitro, apparently reduced the phosphorylation at Ser-293, whereas PD98059, a potent MAPK/ERK inhibitor, did not. High frequency stimulation of mouse hippocampal slices reduced the phosphorylation at Ser-293, albeit in the midst of MAPK/ERK activation. The endophilin binding in vitro was inhibited by phosphorylating Amph I with Mnb/Dyrk1A. However, phosphorylation at Ser-293 did not appear to alter cellular distribution patterns of the protein. Our results suggest that Mnb/Dyrk1A, not MAPK/ERK, is responsible for in vivo phosphorylation of Amph I at Ser-293 and that phosphorylation changes the recruitment of endophilin at the endocytic sites.

Cell-type-specific pathways of neurotensin endocytosis

The neurotensin receptor subtype 1 (NTS1) is a G-protein-coupled receptor (GPCR) mediating a large number of central and peripheral effects of neurotensin. Upon stimulation, NTS1 is rapidly internalized and targeted to lysosomes. This process depends on the interaction of the phosphorylated receptor with beta-arrestin. Little is known about other accessory endocytic proteins potentially involved. Here, we investigated the involvement of dynamin, amphiphysin, and intersectin in the internalization of NTS1 receptor-ligand complexes in transfected COS-7 and HEK 293 cells, by using the transferrin receptor as an internal control for the constitutive endocytic pathway. We found that NTS1 endocytosis was not only arrestin-dependent, but also dynamin-dependent in both COS-7 and HEK 293 cells, whereas internalization of the transferrin receptor was independent of arrestin but required dynamin. Overexpression of the SH3 domain of amphiphysin II had no effect on receptor internalization in either cell type. By contrast, overexpression of full-length intersectin or of its SH3 domain (but not of its EH domain) inhibited NTS1 internalization in COS-7 but not in HEK 293 cells. This difference between COS-7 and HEK 293 cells was not attributable to differences in endogenous intersectin levels between the two cell lines. Indeed, the same constructs inhibited transferrin endocytosis equally well in COS-7 and HEK 293 cells. However, immunogold electron microscopy revealed that internalized NTS1 receptors were associated with clathrin-coated pits in COS-7 cells but with smooth vesicles in HEK 293 cells, suggesting that NTS1 internalization proceeds via different endocytic pathways in these two cell types.

Kinetics of Src Homology 3 Domain Association with the Proline-rich Domain of Dynamins: SPECIFICITY, OCCLUSION, AND THE EFFECTS OF PHOSPHORYLATION

Dynamin function is mediated in part through association of its proline-rich domain (PRD) with the Src homology 3 (SH3) domains of several putative binding proteins. To assess the specificity and kinetics of this process, we undertook surface plasmon resonance studies of the interaction between isolated PRDs of dynamin-1 and -2 and several purified SH3 domains. Glutathione S-transferase-linked SH3 domains bound with high affinity (K(D) approximately 10 nm to 1 microm) to both dynamin-1 and -2. The simplest interaction appeared to take place with the amphiphysin-SH3 domain; this bound to a single high affinity site (K(D) approximately 10 nm) in the C terminus of dynamin-1 PRD, as predicted by previous studies. Binding to the dynamin-2 PRD was also monophasic but with a slightly lower affinity (K(D) approximately 25 nm). Endophilin-SH3 binding to both dynamin-1 and -2 PRDs was biphasic, with one high affinity site (K(D) approximately 14 nm) in the N terminus of the PRD and another lower affinity site (K(D) approximately 60 nm) in the C terminus of dynamin-1. The N-terminal site in dynamin-2 PRD had a 10-fold lower affinity for endophilin-SH3. Preloading of dynamin-1 PRD with the amphiphysin-SH3 domain partially occluded binding of the endophilin-SH3 domain, indicating overlap between the binding sites in the C terminus, but endophilin was still able to interact with the high affinity N-terminal site. This shows that more than one SH3 domain can simultaneously bind to the PRD and suggests that competition probably occurs in vivo between different SH3-containing proteins for the limited number of PXXP motifs. Endophilin-SH3 binding to the high affinity site was disrupted when dynamin-1 PRD was phosphorylated with Cdk5, indicating that this site overlaps the phosphorylation sites, but amphiphysin-SH3 binding was unaffected. Other SH3 domains showed similarly complex binding characteristics, and substantial differences were noted between the PRDs from dynamin-1 and -2. For example, SH3 domains from c-Src, Grb2, and intersectin bound only to the C-terminal half of dynamin-2 PRD but to both the N- and C-terminal portions of dynamin-1 PRD. Thus, differential binding of SH3 domain-containing proteins to dynamin-1 and -2 may contribute to the distinct functions performed by these isoforms.

A putative role for intramolecular regulatory mechanisms in the adaptor function of amphiphysin in endocytosis

Amphiphysin 1 is a brain-specific protein enriched at the synapse and a major binding partner of several components of the clathrin-mediated endocytic machinery (Proc Natl Acad Sci USA 93 (1996) 331). It interacts with clathrin-coat proteins, dynamin, and membranes (Nat Cell Biol 1 (1999) 33; JBC). A role of amphiphysin in synaptic vesicle recycling is supported by both acute and chronic perturbation studies (Science 276 (1997) 259; Neuron 33 (2002) 789). Here we show that amphiphysin directly stimulates clathrin recruitment onto liposomes in an in vitro assay. Amphiphysin-dependent clathrin-coat recruitment is enhanced by the interaction of amphiphysin with dynamin. We also show that the amphiphysin SH3 domain binds full-length amphiphysin, likely via an internal poly-proline region, and that clathrin recruitment onto liposomes by amphiphysin is enhanced in the presence of the isolated amphiphysin SH3 domain. Expression of a mutant amphiphysin harboring two amino acid substitutions in the SH3 domain, and therefore unable to bind proline-containing motifs, induces an accumulation of large intracellular aggregates including amphiphysin, clathrin, AP-2, and other endocytic proteins, as well as a concomitant block of transferrin endocytosis. Thus, putative intramolecular interactions between the amphiphysin COOH-terminal SH3 domain and its internal poly-proline region may regulate clathrin recruitment onto membranes.

Human rhinovirus type 2 is internalized by clathrin-mediated endocytosis.

Using several approaches, we investigated the importance of clathrin-mediated endocytosis in the uptake of human rhinovirus serotype 2 (HRV2). By means of confocal immunofluorescence microscopy, we show that K(+) depletion strongly reduces HRV2 internalization. Viral uptake was also substantially reduced by extraction of cholesterol from the plasma membrane with methyl-beta-cyclodextrin, which can inhibit clathrin-mediated endocytosis. In accordance with these data, overexpression of dynamin K44A in HeLa cells prevented HRV2 internalization, as judged by confocal immunofluorescence microscopy, and strongly reduced infection. We also demonstrate that HRV2 bound to the surface of HeLa cells is localized in coated pits but not in caveolae. Finally, transient overexpression of the specific dominant-negative inhibitors of clathrin-mediated endocytosis, the SH3 domain of amphiphysin and the C-terminal domain of AP180, potently inhibited internalization of HRV2. Taken together, these results indicate that HRV2 uses clathrin-mediated endocytosis to infect cells.

Sorting nexin 4 and amphiphysin 2, a new partnership between endocytosis and intracellular trafficking.

Endocytosis is a regulated physiological process by which membrane receptors and their extracellular ligands are internalized. After internalization, they enter the endosomal trafficking pathway for sorting and processing. Amphiphysins consist of a family of proteins conserved throughout evolution that are crucial elements of the endocytosis machinery in mammalian cells. They act as adaptors for a series of proteins important for the endocytic process, such as dynamin. In order to improve our knowledge of amphiphysin function, we performed a two-hybrid screen with the N-terminal part of murine amphiphysin 2 (residues 1-304). One of the interacting clones corresponded to sorting nexin 4 (SNX4), a member of the SNX family of proteins which are suspected to regulate vesicular trafficking. This interaction was confirmed in vivo by co-immunoprecipitation. Immunofluorescence analysis revealed that amphiphysin 2 might bind reticulo-vesicular structures present throughout the cell body and be associated with SNX4 on these structures. In an endocytosis assay, overexpressed C-terminal or full-length SNX4 was able to inhibit transferrin receptor endocytosis as efficiently as the SH3 domain of amphiphysin 2. At lower levels of expression, SNX4 colocalized with transferrin-containing vesicles, some of which were also positive for amphiphysin 2. These results indicate that SNX4 may be part of the endocytic machinery or, alternatively, that SNX4 may associate with key elements of endocytosis such as amphiphysin 2 and sequester them when overexpressed. The presence of amphiphysin 2 on intracellular vesicles and its interplay with SNX4, which is likely to take part in intracellular trafficking, suggest that amphiphysin 2 is not only a regulator of the early steps of endocytosis. It could also play a role at the surface of the endocytic vesicle that has just been formed and of the future endosomes, in order to regulate intracellular trafficking.

Dynamin regulates focal exocytosis in phagocytosing macrophages.

Phagocytosis in macrophages is thought to involve insertion of cytoplasmic vesicles at sites of membrane expansion before particle ingestion ("focal" exocytosis). Capacitance (Cm) measurements of cell surface area were biphasic, with an initial rise indicative of exocytosis followed by a fall upon phagocytosis. Unlike other types of regulated exocytosis, the Cm rise was insensitive to intracellular Ca2+, but was inhibited by guanosine 5'-O-(2-thio)diphosphate. Particle uptake, but not Cm rise, was affected by phosphatidylinositol 3-kinase inhibitors. Inhibition of actin polymerization eliminated the Cm rise, suggesting possible coordination between actin polymerization and focal exocytosis. Introduction of anti-pan-dynamin IgG blocked Cm changes, suggesting that dynamin controls focal exocytosis and thereby phagocytosis. Similarly, recombinant glutathione S-transferase*amphiphysin-SH3 domain, but not a mutated form that cannot bind to dynamin, inhibited both focal exocytosis and phagocytosis. Immunochemical analysis of endogenous dynamin distribution in macrophages revealed a substantial particulate pool, some of which localized to a presumptive endosomal compartment. Expression of enhanced green fluorescent protein*dynamin-2 showed a motile dynamin pool, a fraction of which migrated toward and within the phagosomal cup. These results suggest that dynamin is involved in the production and/or movement of vesicles from an intracellular organelle to the cell surface to support membrane expansion around the engulfed particle.

Specific interactions of neuronal focal adhesion kinase isoforms with Src kinases and amphiphysin.

Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase that activates Src family kinases via SH2- and SH3-mediated interactions. Specific FAK isoforms (FAK+), responsive to depolarization and neurotransmitters, are enriched in neurons. We analyzed the interactions of endogenous FAK+ and recombinant FAK+ isoforms containing amino acid insertions (boxes 6,7,28) with an array of SH3 domains and the c-Src SH2/SH3 domain tandem. Endogenous FAK+ bound specifically to the SH3 domains of c-Src (but not n-Src), Fyn, Yes, phosphtidylinositol-3 kinase, amphiphysin II, amphiphysin I, phospholipase Cgamma and NH2-terminal Grb2. The inclusion of boxes 6,7 was associated with a significant decrease in the binding of FAK+ to the c-Src and Fyn SH3 domains, and a significant increase in the binding to the Src SH2 domain, as a consequence of the higher phosphorylation of Tyr-397. The novel interaction with the amphiphysin SH3 domain, involving the COOH-terminal proline-rich region of FAK, was confirmed by coimmunoprecipitation of the two proteins and a closely similar response to stimuli affecting the actin cytoskeleton. Moreover, an impairment of endocytosis was observed in synaptosomes after internalization of a proline-rich peptide corresponding to the site of interaction. The data account for the different subcellular distribution of FAK and Src kinases and the specific regulation of the transduction pathways linked to FAK activation in the brain and implicate FAK in the regulation of membrane trafficking in nerve terminals.

The Calcineurin-binding Protein Cain Is a Negative Regulator of Synaptic Vesicle Endocytosis

During neurotransmitter release, exocytosed neurotransmitter vesicles are recycled by endocytosis, which involves the assembly of a complex of endocytic proteins. Assembly of endocytic proteins into a functional complex depends on their dephosphorylation by calcineurin, a calcium-sensitive protein phosphatase and the inhibitory target of immunosuppressive drugs cyclosporin A and FK506. Cain is a recently identified protein inhibitor of calcineurin. We now provide evidence that cain is a component of the endocytic protein complex. The proline-rich region of cain forms a stable association with the SH3 domain of amphiphysin 1. Using a transferrin uptake assay, we found that overexpression of cain in HEK293 cells blocks endocytosis as potently as expression of a dominant negative dynamin 1 construct. The use of other calcineurin inhibitors such as cyclosporin A and FK506 also blocks endocytosis. Since binding of cain to amphiphysin 1 does not affect amphiphysin's interaction with other endocytic proteins, our results suggest that cain negatively regulates synaptic vesicle endocytosis by inhibiting calcineurin activity, rather than sterically interfering with the assembly of the endocytic protein complex.

Expression and purification of dynamin II domains and initial studies on structure and function.

Dynamin II, a large GTP-binding protein, is involved in endocytosis and in vesicle formation at the trans-Golgi network. To further elucidate functions of dynamin II, the pleckstrin homology domain (PHD), the proline-rich domain (PRD), and the C-terminal part of dynamin II (dynamin(500-870)) were expressed in Escherichia coli. The PHD, tagged C-terminally by a (His)(6) peptide, was expressed to 15% of cellular proteins and could be purified on nickel-chelating agarose. On the contrary, the PRD and dynamin(500-870) had to be tagged with a (His)(6) peptide at the N-terminus to bind to nickel-chelating agarose. Additional tagging with the S-peptide, which forms a stable complex with immobilized S-protein, allowed removal of strongly interacting E. coli proteins. Circular dichroic spectra indicate a structured recombinant PHD with a secondary structure content similar to that of the known PHD from dynamin I. The N-terminally tagged, recombinant PRD is unfolded but nevertheless binds specifically to the SH3 domain of amphiphysin II as well as to proteins extracted from rat brain. The described methods are suitable to isolate functionally active domains of dynamin II in sufficient amount and purity for further studies.

Essential Role of Dynamin in Internalization of M2Muscarinic Acetylcholine and Angiotensin AT1AReceptors

Most G protein-coupled receptors (GPCRs), including the M(1) muscarinic acetylcholine receptor (mAChR), internalize in clathrin-coated vesicles, a process that requires dynamin GTPase. The observation that some GPCRs like the M(2) mAChR and the angiotensin AT(1A) receptor (AT(1A)R) internalize irrespective of expression of dominant-negative K44A dynamin has led to the proposal that internalization of these GPCRs is dynamin-independent. Here, we report that, contrary to what is postulated, internalization of M(2) mAChR and AT(1A)R in HEK-293 cells is dynamin-dependent. Expression of N272 dynamin, which lacks the GTP-binding domain, or K535M dynamin, which is not stimulatable by phosphatidylinositol 4, 5-bisphosphate, strongly inhibits internalization of M(1) and M(2) mAChRs and AT(1A)Rs. Expression of kinase-defective K298M c-Src or Y231F,Y597F dynamin (which cannot be phosphorylated by c-Src) reduces M(1) mAChR internalization. Similarly, c-Src inhibitor PP1 as well as the generic tyrosine kinase inhibitor genistein strongly inhibit M(1) mAChR internalization. In contrast, M(2) mAChR internalization is not (or is only slightly) reduced by expression of these constructs or treatment with PP1 or genistein. Thus, dynamin GTPases are not only essential for M(1) mAChR but also for M(2) mAChR and AT(1A)R internalization in HEK-293 cells. Our findings also indicate that dynamin GTPases are differentially regulated by c-Src-mediated tyrosine phosphorylation.

The SH3 Domains of Endophilin and Amphiphysin Bind to the Proline-rich Region of Synaptojanin 1 at Distinct Sites That Display an Unconventional Binding Specificity

The proline-rich domain of synaptojanin 1, a synaptic protein with phosphatidylinositol phosphatase activity, binds to amphiphysin and to a family of recently discovered proteins known as the SH3p4/8/13, the SH3-GL, or the endophilin family. These interactions are mediated by SH3 domains and are believed to play a regulatory role in synaptic vesicle recycling. We have precisely mapped the target peptides on human synaptojanin that are recognized by the SH3 domains of endophilins and amphiphysin and proven that they are distinct. By a combination of different approaches, selection of phage displayed peptide libraries, substitution analyses of peptides synthesized on cellulose membranes, and a peptide scan spanning a 252-residue long synaptojanin fragment, we have concluded that amphiphysin binds to two sites, PIRPSR and PTIPPR, whereas endophilin has a distinct preferred binding site, PKRPPPPR. The comparison of the results obtained by phage display and substitution analysis permitted the identification of proline and arginine at positions 4 and 6 in the PIRPSR and PTIPPR target sequence as the major determinants of the recognition specificity mediated by the SH3 domain of amphiphysin 1. More complex is the structural rationalization of the preferred endophilin ligands where SH3 binding cannot be easily interpreted in the framework of the "classical" type I or type II SH3 binding models. Our results suggest that the binding repertoire of SH3 domains may be more complex than originally predicted.

Perturbation of Dynamin II with an Amphiphysin SH3 Domain Increases GLUT4 Glucose Transporters at the Plasma Membrane in 3T3-L1 Adipocytes: DYNAMIN II PARTICIPATES IN GLUT4 ENDOCYTOSIS

The GLUT4 glucose transporter continuously recycles between the cell surface and an endosomal compartment in adipocytes. Insulin decreases the rate of GLUT4 endocytosis in addition to increasing its exocytosis. Endocytosis of the transporter is thought to occur at least in part via the clathrin-mediated endocytic system. The protein dynamin is involved in the final stages of clathrin-coated vesicle formation. Here we show that the dynamin II isoform is expressed in 3T3-L1 adipocytes and is present in isolated plasma membrane and low density microsomal fractions. Insulin reduced the levels of dynamin II associated with the plasma membrane by about half, raising the possibility that the hormone may reduce GLUT4 endocytosis by removing dynamin from the cell surface. A fusion protein containing the amphiphysin SH3 domain selectively bound dynamin II from 3T3-L1 adipocyte cell lysates. Microinjection of the fusion protein into these cells inhibited transferrin endocytosis and increased the levels of GLUT4 at the cell surface. Glutathione S-transferase alone, the SH3 domains of spectrin and Crk, and a mutated amphiphysin SH3 domain unable to bind dynamin II did not affect GLUT4 distribution. However, a peptide containing the dynamin II sequence that binds amphiphysin increased the surface presence of GLUT4. Moreover, in cells first treated with insulin to externalize GLUT4, the dynamin peptide, but not an unrelated control peptide, inhibited GLUT4 internalization upon insulin removal. These results suggest that interactions of dynamin II with amphiphysin may play an important role in GLUT4 endocytosis. We hypothesize that insulin may reduce GLUT4 endocytosis by regulating the function of dynamin II at the cell surface, as part of the mechanism to increase glucose uptake.

Novel variants of amphiphysin I expressed in the retina

Amphiphysin 1 is a brain-specific protein enriched at the synapse and a major binding partner of several components of the clathrin-mediated endocytic machinery (Proc Natl Acad Sci USA 93 (1996) 331). It interacts with clathrin-coat proteins, dynamin, and membranes (Nat Cell Biol 1 (1999) 33; JBC). A role of amphiphysin in synaptic vesicle recycling is supported by both acute and chronic perturbation studies (Science 276 (1997) 259; Neuron 33 (2002) 789). Here we show that amphiphysin directly stimulates clathrin recruitment onto liposomes in an in vitro assay. Amphiphysin-dependent clathrin-coat recruitment is enhanced by the interaction of amphiphysin with dynamin. We also show that the amphiphysin SH3 domain binds full-length amphiphysin, likely via an internal poly-proline region, and that clathrin recruitment onto liposomes by amphiphysin is enhanced in the presence of the isolated amphiphysin SH3 domain. Expression of a mutant amphiphysin harboring two amino acid substitutions in the SH3 domain, and therefore unable to bind proline-containing motifs, induces an accumulation of large intracellular aggregates including amphiphysin, clathrin, AP-2, and other endocytic proteins, as well as a concomitant block of transferrin endocytosis. Thus, putative intramolecular interactions between the amphiphysin COOH-terminal SH3 domain and its internal poly-proline region may regulate clathrin recruitment onto membranes.