The SH3 Domain of Amphiphysin Binds the Proline-rich Domain of Dynamin at a Single Site That Defines a New SH3 Binding Consensus Sequence

Abstract

Amphiphysin is an SH3 domain-containing neuronal protein that is highly concentrated in nerve terminals where it interacts via its SH3 domain with dynamin I, a GTPase implicated in synaptic vesicle endocytosis. We show here that the SH3 domain of amphiphysin, but not a mutant SH3 domain, bound with high affinity to a single site in the long proline-rich region of human dynamin I, that this site was distinct from the binding sites for other SH3 domains, and that the mutation of two adjacent amino acids in dynamin I was sufficient to abolish binding. The dynamin I sequence critically required for amphiphysin binding (PSRPNR) fits in the novel SH3 binding consensus identified for the SH3 domain of amphiphysin via a combinatorial peptide library approach: PXRPXR(H)R(H). Our data demonstrate that the long proline-rich stretch present in dynamin I contained multiple SH3 domain binding sites that recognize interacting proteins with high specificity.Dynamin I is a neuronal GTPase concentrated in nerve terminals that plays an essential role in synaptic vesicle endocytosis and recycling (for reviews, see Refs. 1 and 2). A temperature-sensitive mutation in the dynamin I gene of Drosophila leads to rapid and massive block of synaptic vesicle endocytosis resulting in a paralytic phenotype (3-5). Ultrastructural studies have shown that dynamin I forms rings at the neck of clathrin-coated pits, and it has been hypothesized that a conformational change of the ring that correlates with GTP hydrolysis represents a key step leading to vesicle fission from the plasmalemma (6, 7). In addition, dynamin I has also been implicated in rapid endocytosis, a form of Ca 2ϩ -triggered endocytosis detectable by capacitance measurement in neuroendocrine cells (8). Dynamin isoforms (dynamin II and dynamin III) are expressed in non-neuronal cells (9 -11) where they are thought to play a general role in clathrin-mediated endocytosis (12, 13).The COOH-terminal region of dynamin I contains a 100-amino acid-long proline-rich domain (14), which undergoes regulation by protein phosphorylation and binds a variety of SH3 domains (13,(15)(16)(17)(18)). An abundant SH3 domain-containing protein, which is a major binding partner for dynamin I in nerve terminals (19), is amphiphysin, the dominant autoantigen in paraneoplastic stiff-man syndrome (20 -22). Amphiphysin is closely colocalized with dynamin I at synapses where, in addition to dynamin I, it binds the presynaptic inositol-5-phosphatase synaptojanin (23). Amphiphysin binds the plasmalemmal clathrin adaptor AP2 via a region distinct from its SH3 domain (19, 24), further supporting an involvement of amphiphysin in endocytosis. In addition, amphiphysin contains regions of similarity to two yeast proteins, Rvs167 and Rvs161 (20,25,26), which genetic studies have shown to be implicated both in endocytosis and in the function of the actin cytoskeleton (26,27).As a premise to further elucidate the functional interconnections between amphiphysin and dynamin I, we investigated regions that are crucial for re...

Beta-amyloid disrupted synaptic vesicle endocytosis in cultured hippocampal neurons

“…P4 blocks the binding of dynamin 1 to amphiphysin and prevents the recruitment of dynamin 1 to the endocytic complex. Biochemical assays showed that this peptide is highly specific and binds with high affinity to a unique site in the dynamin-proline rich domain (Grabs et al 1997;Kittler et al 2000). Treated neurons were then stimulated and used for subcellular fractionation or FM1-43 imaging as described below.…”

“…To obtain more direct evidence of the role of dynamin 1 in the altered synaptic vesicle endocytosis in Aβ-treated neurons, we examined whether inhibition of dynamin 1 would cause a similar membranous build-up of amphiphysinin in cultured hippocampal neurons. To inhibit dynamin 1 activity, we used a dynamin 1 inhibitory peptide (P4) which blocks the binding of dynamin 1 to amphiphysin and prevents the recruitment of dynamin 1 to the endocytic complex (Grabs et al 1997;Kittler et al 2000). When these neurons were treated with 50 μM P4 for 30 minutes prior to stimulation they did not show any change in the amphiphysin membrane-to-cytosol ratio when compared to untreated stimulated neurons (Fig.…”

Direct interaction of Cbl with pTyr 1045 of the EGF receptor (EGFR) is required to sort the EGFR to lysosomes for degradation

“…Self-assembly of dynamin into oligomers activates the GTPasestimulating effect of this domain (Muhlberg et al, 1997;Sever et al, 1999). Through its Proline Rich Domain (PRD), dynamin is further able to interact with a number of SH3 domain containing proteins involved in endocytosis, such as amphiphysin, cortactin, endophilin, Grb2, intersectin and Src (Gout et al, 1993;Miki et al, 1994;Seedorf et al, 1994;Grabs et al, 1997;Ringstad et al, 1997;Foster-Barber and Bishop, 1998;Yamabhai et al, 1998;McNiven et al, 2000). There is increasing evidence that dynamin is involved in the late stages of clathrin-coated vesicle formation, most likely in pinching off of clathrin-coated pits (Kosaka and Ikeda, 1983;Carter et al, 1993;van der Bliek et al, 1993;Damke et al, 1994;Damke et al, 1995;Sever et al, 2000;Damke et al, 2001;Narayanan et al, 2005).…”

The Rsp5 Ubiquitin Ligase Binds to and Ubiquitinates Members of the Yeast CIN85-Endophilin Complex, Sla1-Rvs167

“…A yeast lysate containing HA-Inp52 was prepared as previously described (41), except that the lysis buffer contained 3 mg/ml bovine serum albumin and 1% Nonidet P-40. In addition, beads with immobilized His 6 -tagged polypeptides were preincubated in buffer containing 1 mg/ml bovine serum albumin in Tris-buffered saline, pH 7.5, for 1 h at 4°C.…”