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

Abstract

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...

The amphiphysin family of proteins and their role in endocytosis at the synapse

“…How accurate is this hypothesis in the context of the amphiphysins? The second amphiphysin gene in mammals, amphiphysin 2 (of which various splice variants have been alternatively named BIN-1, SH3P9, BRAMP-2 or ALP-1) has now been cloned by eight independent groups 18,19,[28][29][30][31][32] . The largest isoform, a 95kDa protein, is by far the major type present in the brain, and appears to be brain-specific.…”

“…The largest isoform, a 95kDa protein, is by far the major type present in the brain, and appears to be brain-specific. However, the fact that there are smaller isotypes expressed ubiquitously 19,30 , some in cell type-specific patterns 18 , suggests that the amphiphysin family is indeed capable of acting in more general clathrin-mediated endocytosis outside of the brain.…”

“…An important factor in the formation of clathrin-coated pits is the plasma-membrane adaptor complex AP-2, a heterotetrameric complex that has recently been shown to interact with both Amph1 and Amph2 via its ␣ subunit 19,30,37 . This is mediated through a region that is distinct from the SH3 domain of amphiphysin 20 , and indeed, AP-2 and dynamin can interact with Amph1 simultaneously 22 .…”

Tandem Arrangement of the Clathrin and AP-2 Binding Domains in Amphiphysin 1 and Disruption of Clathrin Coat Function by Amphiphysin Fragments Comprising These Sites

“…Yeast homologues of amphiphysin, Rvs167 and Rvs161, have been implicated in endocytosis, actin function, and signaling (5,6). In mammals, both amphiphysin 1 and 2 are highly expressed in the nervous system, where they are concentrated at synapses and further concentrated on endocytic intermediates implicated in synaptic vesicle recycling (7)(8)(9)(10)(11). Amphiphysin 1 and 2 have a similar structure that includes several main domains.…”

Inhibition of Phospholipase D by Amphiphysins

“…Subsequently, AmphII (of which various splice variants have been named BIN-1, SH3P9, BRAMP-2, or ALP) was cloned by several laboratories (32)(33)(34). The largest isoform of AmphII, comprising 588 amino acids, appears to be brain-specific, whereas smaller isoforms show a widespread tissue distribution.…”