In this article we have investigated the properties of Amph2, in the possibility that it might represent an isoform functionally distinct from Amph1, perhaps participitating in endocytic events other than synaptic vesicle recycling at the plasma membrane. Instead, we find that the subcellular distribution and phosphorylation and membrane association properties of both Amphs closely parallel each other. This suggests that the two proteins act in concert, and indeed, coexpression of the two isoforms in COS cells or in bacteria is sufficient to generate a stable equimolar complex. Cross-linking indicates that this complex exists in vivo as a 220- to 250-kDa heterodimer. Furthermore, the dimer appears to be the predominant form of Amph in the brain, where it can be coimmunoprecipitated with multiple molecules of dynamin I and, therefore, is the likely form of Amph that drives the recruitment of dynamin to sites of clathrin-mediated endocytosis. It is interesting that Amph1, Amph2, and dynamin I are rapidly dephosphorylated in parallel upon nerve-terminal stimulation. On repolarization they are all rephosphorylated by the same kinase, PKC, further supporting the idea that they might function as a complex.
Two Amph homologues, rvs161
, have been identified in Saccharomyces cerevisiae
(David et al., 1994
; Munn et al., 1995
; Sivadon et al., 1995
). Knocking out either of the rvs
genes leads to the full endocytosis-defective phenotype characteristic of the double mutant. Furthermore, both mutant strains are suppressed by the same set of SUR genes (Desfarges et al., 1993
). This has led to the hypothesis that the two proteins function as a complex, possibly a heterodimer, at least in yeast (Bauer et al., 1993
). Although sharing only weak sequence homology to the yeast proteins, Amph1 and Amph2 also form heterodimers, and in this respect at least, the heterodimer appears to have been conserved throughout evolution.
Whether or not the heterodimer has been conserved in function is less clear. The in vivo transferrin uptake assay clearly shows that, although overexpression of either Amph1 or Amph2 alone blocks endocytosis in COS cells, formation of the Amph1–Amph2 heterodimer rescues this defect. This suggests that the heterodimer has a crucial function not fulfilled by the separate Amphs. What is this function? Although Amph1 and Amph2 separately bind dynamin in vitro, the brain heterodimer can interact with multiple dynamin molecules simultaneously and, therefore, bind more on a molar basis than either Amph1 or Amph2. This would imply that both SH3 domains in the heterodimer are accessible to interact with separate dynamin molecules. In synaptic vesicle endocytosis, Amph heterodimers localized to the plasma membrane (probably via an interaction with the AP-2 adaptor complex; Wang et al., 1995
; David et al., 1996
) would allow for efficient recruitment of multiple dynamin molecules to the coated pit. By generating a high local concentration of dynamin molecules in this way, Amph could potentially act as a template for the nucleation of dynamin oligomers, aiding the assembly of dynamin into rings at the neck of the constricted coated pit (Figure ). Although this hypothesis remains to be tested, our experiments showing that the heterodimer (but not either isolated Amph SH3 domain) accelerates dynamin’s GTPase activity in vitro lends support to this model. GTP hydrolysis by dynamin is thought to be a necessary prerequisite for the concerted conformational change in the oligomer that pinches off the vesicle (De Camilli et al., 1995
). If Amph has an important role in this cycle, it is likely that the GTPase activation is tightly controlled; GTP hydrolysis in the cytosol would be premature. To be physiological, Amph should only activate GTP hydrolysis on dynamin when it is assembled at the collar of the coated pit. In vivo, therefore, it is important not to rule out other factors that may come into play as well as Amph.
Figure 9 Hypothetical model for the action of the Amph heterodimer in dynamin recruitment. The SH3 domains of both isoforms are accessible in the dimer to interact with separate dynamin molecules, which are brought into close proximity, catalyzing the formation (more ...)
Why are there so many splice variants of Amph2? We have identified six different alternatively spliced forms; at least some of these are present in peripheral tissues and, therefore, could have more generalized roles. It is interesting that both Amph2–3 and Amph2–4 lack the C-terminal SH3 domain and in this respect resemble the domain structure of the yeast homologue rvs161. The yeast heterodimer, lacking the two SH3 domains characteristic of the rat brain heterodimer we have characterized, would probably have a different function. Such a dimer would not be able to recruit multiple dynamin molecules. It is possible, then, that the brain heterodimer is a feature of the specialized pathway of synaptic vesicle uptake and reflects the need for rapid or efficient dynamin recruitment to endocytosing zones of nerve terminals. Yeast, which does not require such rapid membrane uptake, could cope with just one SH3 domain. However, the yeast dynamin homologue interacting with the Rvs167 protein remains to be identified, and so endocytosis in this organism could, in theory at least, act by a different mechanism entirely.
Particularly intriguing is the possibility that a collection of Amph heterodimers could exist, each with slightly different binding specificities. In this context, we have preliminary data suggesting that Amph2–6, and possibly other splice variants, can heterodimerize with Amph1. The transcription factors Fos and Jun exist in different combinations of dimeric forms, each of which display different DNA-binding specificities. It is possible that a repertoire of different Amph heterodimers could greatly expand the potential for regulation of clathrin-mediated uptake processes.
Although much evidence indicates that the Amph family takes part in endocytosis at the plasma membrane, a second distinct role for one of the Amph2 splice variants, Amph2–7, has been suggested. This isoform has been recently isolated as the nuclear Myc-interacting protein BIN1 (Sakamuro et al., 1996
). Loss of BIN1 mRNA was frequently observed in tumor cell lines, and BIN1 prevented tumorigenesis by Ras in a cooperativity assay. Thus, these data led Sakamuro et al. (1996)
to suggest that BIN1 is a tumor suppressor. Although we found a partial clone encoding a nuclear localization signal, this was not predominant in the cDNA library. We have no other data to support a different function for Amph2, but it is possible that this gene product, depending on how it is spliced and, therefore, to which compartment it is targeted, has the capacity to play two diverse roles in the cell. Proenkephalin, normally a cytoplasmic protein that is destined to be secreted, has also been found to have a possibly distinct function in the nucleus (Bottger and Spruce, 1995
). These authors hypothesized that the two diverse roles of the protein may reflect a molecular economy in the process of evolution, such that “gene sharing” has been adopted.
The heterodimer appears to be the main form of Amph in the brain, and a combination of in vitro and in vivo data suggest it may have an important widespread role in clathrin-mediated endocytosis not fulfilled by either isoform alone.