Growth factor-induced binding of dynamin to signal transduction proteins involves sorting to distinct and separate proline-rich dynamin sequences.

250

220

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...

Section: Discussionsupporting

confidence: 92%

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

Grabs

Slepnev

Songyang

et al. 1997

250

220

“…The interaction with dynamin is direct and likely to rely on the SH3 domain of amphiphysin 2 on one side and on a proline-rich sequence of dynamin on the other side. This conclusion is based on molecular interactions studied on a BIAcore biosensor where a proline-rich peptide of dynamin I corresponding to one of the previously described Grb2-binding sites is partially inhibitory (9,16). This suggests that BRAMP2 interacts at least in part with this proline-rich sequence.…”

Section: Resultsmentioning

confidence: 83%

A New Member of the Amphiphysin Family Connecting Endocytosis and Signal Transduction Pathways

Leprince¹,

Romero²,

Cussac³

et al. 1997

Src homology 3 (SH3) domains are conserved modules which participate in protein interaction by recognizing proline-rich motifs on target molecules. To identify new SH3-containing proteins, we performed a two-hybrid screen with a proline-rich region of human SOS-1. One of the specific SOS-1 interacting clones that were isolated from a mouse brain cDNA library defines a new protein that was named amphiphysin 2 because of its homology to the previously reported amphiphysin. Amphiphysin 2 is expressed in a number of mouse tissues through multiple RNA transcripts. Here, we report the amino acid sequence of a brain form of amphiphysin 2 (BRAMP2) encoded by a 2.5-kilobase mRNA. BRAMP2 associates in vitro with elements of the endocytosis machinery such as ␣-adaptin and dynamin. On a biosensor surface, the BRAMP2/dynamin interaction appeared to be direct and partly dependent on a proline-rich sequence of dynamin. Association with dynamin was also observed in PC12 cells after cell stimulation with nerve growth factor, suggesting that amphiphysin 2 may be connected to receptor-dependent signaling pathways. This hypothesis is strengthened by the ability of BRAMP2 to interact with the p21 ras exchange factor SOS, in vitro, as a possible point of interconnection between the endocytic and signaling pathways.A limited set of protein modules mediates molecular interactions and underlies the diversity of intracellular signaling pathways. Among these modules, Src homology 3 (SH3) 1 domains were first identified as non-catalytic regions of 55-70 AA, present in signaling and cytoskeletal proteins (1). A common feature of SH3 domains is their ability to bind proline-rich sequences on target molecules. A SH3 domain is composed of a hydrophobic binding pocket with finely positioned aromatic residues and charged loops outside of this pocket that determine the specificity of the interaction with proline-rich motifs. Phage display or peptide library screenings with individual SH3 domains demonstrated the absence of a strict exclusivity for SH3 and proline-rich partners (2, 3). This feature may be the basis for the identification of new SH3-containing proteins.Human SOS-1 C terminus contains a multitude of intermingled proline-rich motifs. It was used as a "pseudo-degenerated" bait in search for new SH3 domains in a two-hybrid screen. Several SH3 domains were isolated, and one of them allowed us to identify a new protein, homologous to, but different from, amphiphysin (4), that was named amphiphysin 2. Amphiphysin is a strictly neuronal protein that plays a crucial role in the endocytosis of synaptic vesicles (5). A current hypothesis suggests that a ubiquitous homologue involved in general endocytosis should exist. We show here that this hypothesis is correct and incomplete. Amphiphysin 2 is expressed under several forms: a 2.0-kb transcript which is expressed almost ubiquitously and a 2.5-kb transcript that is brain-specific. The latter form was further characterized in vitro and in vivo. We show that the brain form of amphiphysin 2 (BRA...

“…Further correlative evidence is provided by the binding affinity detected between eNOS and dyn-2 PRD, which is relatively similar to that observed between eNOS and the dyn-2 full-length protein. Detection of PRD as the eNOS binding region of dyn-2 is not entirely unexpected because dyn-2 PRD is responsible for many of the protein interactions with which dynamins are associated, including AP-2, Grb2, phospholipase C␥, and phosphatidylinositol 3-kinase (24,25,28). Proline-rich sequences are a common ligand preference for a variety of protein interaction domains (42), and many dynamin-associ- FIG.…”

Section: Discussionmentioning

confidence: 99%

“…Because many dynamin-associated proteins bind by virtue of an SH3 domain-PRD interaction (24,25,28) and the eNOS sequence that binds dyn-2 PRD contains an SH3-like region at amino acids 767-823 (Fig. 2E), we next sought to determine whether dyn-2 PRD binding with eNOS could be competed by other SH3 domain-containing proteins.…”

Section: Dyn-2 Prd and Enosmentioning

confidence: 99%

“…PHD and PRD regions are prominently implicated in regulatory binding interactions. More specifically, PHD binds phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 4-phosphate, whereas PRD binds specific proteins, often notable for the presence of an SH3 domain, including phospholipase C␥, Nck, AP-2, Grb2, and phosphatidylinositol 3-kinase (23)(24)(25)(26)(27)(28)(29)(30).…”

mentioning

confidence: 99%

See 1 more Smart Citation

The Proline-rich Domain of Dynamin-2 Is Responsible for Dynamin-dependent in Vitro Potentiation of Endothelial Nitric-oxide Synthase Activity via Selective Effects on Reductase Domain Function

Cao

Yao

Shah

2003

Here we demonstrate, using purified proteins, that this occurs through a selective influence of the dyn-2 proline-rich domain (dyn-2 PRD) on the eNOS reductase domain. In vitro studies demonstrate that dyn-2 PRD fused with glutathione S-transferase (GST) binds recombinant eNOS protein specifically and with binding kinetics comparable with that observed between dyn-2 fulllength and eNOS. Additionally, GST-dyn-2 PRD binds the in vitro transcribed 35 S-eNOS reductase domain but not the 35 S-eNOS oxygenase domain. Furthermore GSTdyn-2 PRD binds a 35 S-labeled eNOS reductase domain fragment (amino acids 645-850) that partially overlaps with the FAD binding domain of eNOS. A recombinant form of the SH3-containing protein Fyn competes the binding of recombinant eNOS protein with dyn-2 PRD, thereby implicating the SH3-like region contained within this reductase domain fragment as the dyn-2 binding region. Mammalian two-hybrid screen corroborates these interactions in cells as well. Functional studies demonstrate that dyn-2 PRD selectively potentiates eNOS activity in a concentration-dependent manner in an order of magnitude similar to that observed with dyn-2 full-length and in a manner that requires calmodulin. Although dyn-2 PRD does not influence eNOS oxygenase domain function or ferricyanide reduction, it does potentiate the ability of recombinant eNOS to reduce cytochrome c, supporting an influence of dyn-2 PRD on electron transfer between FAD and FMN. (These data indicate that the binding domains of dyn-2 and eNOS reside within the dyn-2 PRD domain and the FAD binding region of the eNOS reductase domains, respectively, and that dyn-2 PRD is sufficient to mediate dyn-2-dependent potentiation of eNOS activity, at least in part, by potentiating electron transfer.)Endothelial nitric-oxide synthase (eNOS) 1 is a membraneassociated protein that catalyzes the conversion of L-arginine to L-citrulline and nitric oxide (NO) (1). eNOS function is regulated in part by post-translational mechanisms including acylation, phosphorylation, and protein interactions (2-7). Indeed, specific proteins have been identified which interact with eNOS, thereby regulating enzyme function, including the large GTPase, dynamin-2 (dyn-2) (8 -12). With regard to the latter, it has been demonstrated that dyn-2 specifically binds eNOS in a direct manner and potentiates the ability of eNOS to convert L-arginine to L-citrulline in a concentration-dependent manner (12). However, delineation of binding domains and the mechanism of activation remain unexplored.eNOS is a bi-domain enzyme that requires a number of cofactors and substrates to generate NO optimally via an orchestrated electron transfer mechanism (1). An oxygenase domain (amino acids 1-491) contains binding sites for tetrahydrobiopterin, heme, and L-arginine, and a reductase domain (amino acids 492-1205) contains binding sites for calmodulin, FMN, FAD, and NADPH (1). Electron flux is initiated at the reductase domain where NADPH-derived electrons are transferred sequentially through the boun...