Concurrent specific immunological detection of both primate and rodent forms of the guanine nucleotide binding protein G11 alpha following their coexpression.
The phosphoinositidase C-linked G proteins Gq alpha and G11 alpha are highly similar and comigrate in 10% (w/v) acrylamide SDS-PAGE. Antisera generated against regions common between these G proteins thus detect a composite of the two polypeptides following resolution in such gels. Using SDS-PAGE conditions which allow resolution of Gq alpha and G11 alpha in rodent brain and neuroblastoma cell lines it was observed that primate frontal cortex and neuroblastoma cell lines did not express a polypeptide which comigrated with rodent G11 alpha. Species diversity in G-protein sequences is extremely limited; however, immunoblotting primate cells and frontal cortex with a G11 alpha-specific antiserum demonstrated this to be due to a difference in mobility of rodent and primate G11 alpha under these conditions rather than lack of expression of G11 alpha by primates. A cDNA encoding mouse G11 alpha was transiently expressed in monkey COS-1 cells and membranes from these cells were immunoblotted with antisera able to identify primate and rodent G11 alpha equally, following SDS-PAGE under the resolving conditions. Both mouse and monkey G11 alpha could be detected concurrently and unambiguously following transfection. This is the first demonstration that species variants of the same G protein expressed in a single cell can be detected simultaneously.
Kim
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Department of Biochemistry, University of Glasgow, UK.
Milligan
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D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002540:Cerebral Cortex / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000032:analysis* / Q000302:isolation & purification / Q000378:metabolism; D015870:Gene Expression; D006168:Guinea Pigs; D000882:Haplorhini; D006801:Humans; D051379:Mice; D010215:Papio; D051381:Rats; D013552:Swine; D014162:Transfection; D014407:Tumor Cells, Cultured
D019204:GTP-Binding Proteins