Insulin regulation of a novel WD-40 repeat protein in adipocytes

A 400 bp PCR product generated with degenerate primers derived from the glu agon-like peptide-1 receptor was used to screen a rat skeletal muscle cDNA library. The predicted amino acid sequence oft he 978 bp open reading frame has a predicted M-r of 35 804, an estimated isoelectric point (pI) of 5.31 and contains seven WD-40 repeats, which are common to G-protein beta subun its (G beta). Although chemically and structurally similar to G beta subuni ts, the predicted amino acid sequence, when compared with the previously cl oned G beta isoforms, was found to be only 31-41% similar and thus was name d G beta -like (G betaL, 'Gable'). Western blotting of whole-cell lysates a nd immunoprecipitates of membrane and cytosolic fractions of HEK 293 cells stably overexpressing a carboxy-terminal His-tagged G betaL indicates that the protein is cytosolic and that it migrates at 42 kDa. A 4 kb transcript was detected in all tissues surveyed by northern blotting; however, an addi tional 2 kb transcript was detected in testis. Expression of G betaL mRNA w as highest in the brain and testis, followed by lung, heart, kidney, skelet al muscle, spleen and liver. In addition, reverse transcriptase/PCR showed that several other tissues and cell lines express G betaL. The ubiquitous n ature of the tissue expression pattern of G betaL is similar to thar of the insulin receptor, which suggests that insulin may influence G betaL expres sion. Indeed, G betaL protein and mRNA levels, in fully differentiated 3T3- L1 adipocytes, were upregulated by insulin in a concentration-dependent fas hion. These changes were highly sensitive to insulin stimulation, being min imally affected by doses as low as 0.1 nM and maximally elevated by 1 nM do ses. These data suggest that insulin regulates G betaL production and imply that some of the actions of insulin may be mediated, in part, by this nove l intracellular protein.