Abstract
Cbl-associated protein (CAP) is an adaptor protein that plays important roles in both signal transduction and cytoskeleton rearrangement. Alternative splicing of the gene SORBS1 results in multiple isoforms of CAP protein. We report here the cloning of 3 new CAP isoforms, CAP2, CAP3, and CAP4, from mouse adipose tissue. RT-PCR analyses reveal that the isoform mRNAs are differentially expressed. CAP2, CAP3, and CAP4 contain a coiled-coil domain. In addition, CAP4 contains a proline-rich region, part of which exists in CAP3. Coimmunoprecipitation experiments show that CAP4 forms a homodimeric complex. While these new isoforms similarly interact with Cbl, they exhibit varied binding specificity toward vinculin. In contrast to CAP1 and CAP2, CAP4 does not interact with vinculin, and CAP3 binds with low affinity. Immunofluorescence analysis demonstrates differential subcellular localization of Myc-tagged CAP isoforms in 3T3-L1 adipocytes. These results suggest that these new isoforms of CAP might play different signaling roles.
Highlights
Cbl-associated protein (CAP) is a widely expressed, multifunctional adaptor protein involved in intracellular signaling [1,2]
Cloning of CAP Splicing Isoforms Total RNA was extracted from mouse white adipose tissue (WAT), and RT-PCR was performed to amplify the full-length CAP variants, using a primer set flanking the coding region of the originally cloned CAP, which is designated CAP1
CAP has been suggested to play a role in cytoskeleton rearrangement, especially the formation of focal adhesions, which may involve its binding with vinculin
Summary
Cbl-associated protein (CAP) is a widely expressed, multifunctional adaptor protein involved in intracellular signaling [1,2]. CAP belongs to the sorbin homology (SoHo) family of proteins, including vinexin and ArgBP2, which contain 1 or 2 SoHo domains in their amino terminus and 3 adjacent SH3 domains in their carboxyl terminus [3]. Accumulating evidence suggests that the proteins in this family are involved in the organization of both signal transduction and the cytoskeleton. Cbl constitutively associates with CAP, and upon insulin stimulation, both are recruited to the insulin receptor by the adapter protein APS [4]. The Cbl-CAP complex accumulates in lipid raft subdomains in the plasma membrane through the interaction of CAP with the raft-associated protein, flotillin [3]. Phosphorylated Cbl recruits CrkII to the lipid raft, which initiates downstream signaling [5]
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