Related Experiment Videos
Meltrin alpha cytoplasmic domain interacts with SH3 domains of Src and Grb2 and is phosphorylated by v-Src
Abstract:
Meltrin alpha/ADAM12 is a member of the ADAM/MDC family proteins characterized by the presence of metalloprotease and disintegrin domains. This protein also contains a single transmembrane domain and a relatively long cytoplasmic domain containing several proline-rich sequences. These sequences are compatible with the consensus sequences for binding the Src homology 3 (SH3) domains. To determine whether the proline-rich sequences interact with SH3 domains in several proteins, binding of recombinant SH3 domains to the meltrin alpha cytoplasmic domain was analysed by pull-down assays. The SH3 domains of Src and Yes bound strongly, but that of Abl or phosphatidylinositol 3-kinase p85 subunit did not. Full-length Grb2/Ash bound strongly, whereas its N-terminal SH3 domain alone did less strongly. Src and Grb2 in bovine brain extracts also bound to meltrin alpha cytoplasmic domain on affinity resin. Furthermore, immunoprecipitation with a monoclonal antibody to meltrin alpha resulted in coprecipitation of Src and Grb2 with meltrin alpha in cell extracts, suggesting that Src and Grb2 are associated in vivo with meltrin alpha cytoplasmic domain. This notion was also supported by the findings that exogenously expressed meltrin cytoplasmic domain coexisted with Src and Grb2 on the membrane ruffles. The C-terminal Tyr901 of meltrin alpha was phosphorylated both in vitro and in cultured cells by v-Src. These results may imply that meltrin alpha cytoplasmic domain is involved in a signal transduction for some biological function through the interaction with SH3-containing proteins.
Insights
Meltrin alpha (ADAM12) interacts with Src and Grb2 proteins via its cytoplasmic domain. This interaction suggests meltrin alpha
Area of Science:
- Cell biology
- Molecular biology
- Protein interactions
Background:
- Meltrin alpha (ADAM12) is a transmembrane protein with metalloprotease and disintegrin domains.
- Its cytoplasmic domain contains proline-rich sequences, suggesting potential interactions with SH3 domains.
- SH3 domains are crucial for mediating protein-protein interactions in signal transduction pathways.
Purpose of the Study:
- To investigate the interaction between the meltrin alpha cytoplasmic domain and SH3 domains of various proteins.
- To determine if meltrin alpha associates with SH3-containing proteins in vivo.
Main Methods:
- Pull-down assays using recombinant SH3 domains and meltrin alpha cytoplasmic domain.
- Affinity resin binding assays with bovine brain extracts.
- Immunoprecipitation using anti-meltrin alpha antibody.
- Analysis of co-localization of exogenously expressed meltrin alpha and Src/Grb2.
- In vitro and in cell-based phosphorylation assays.
Main Results:
- The SH3 domains of Src and Yes, as well as full-length Grb2/Ash, bound strongly to the meltrin alpha cytoplasmic domain.
- Src and Grb2 were found to associate with meltrin alpha in bovine brain extracts and cell extracts.
- Exogenously expressed meltrin alpha cytoplasmic domain co-localized with Src and Grb2 on membrane ruffles.
- The C-terminal Tyr901 of meltrin alpha was phosphorylated by v-Src.
Conclusions:
- Meltrin alpha cytoplasmic domain interacts with SH3-containing proteins like Src and Grb2.
- These interactions suggest a role for meltrin alpha in signal transduction pathways.
- Meltrin alpha may be involved in regulating cellular functions through its association with Src and Grb2.