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

A 400 bp PCR product generated with degenerate primers derived from the glucagon-like peptide-1 receptor was used to screen a rat skeletal muscle cDNA library. The predicted amino acid sequence of the 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 subunits (Gβ). Although chemically and structurally similar to Gβ subunits, the predicted amino acid sequence, when compared with the previously cloned Gβ isoforms, was found to be only 31-41% similar and thus was named Gβ-like (GβL, 'Gable'). Western blotting of whole-cell lysates and immunoprecipitates of membrane and cytosolic fractions of HEK 293 cells stably overexpressing a carboxy-terminal His-tagged GβL 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 additional 2 kb transcript was detected in testis. Expression of GβL mRNA was highest in the brain and testis, followed by lung, heart, kidney, skeletal muscle, spleen and liver. In addition, reverse transcriptase/PCR showed that several other tissues and cell lines express GβL. The ubiquitous nature of the tissue expression pattern of GβL is similar to that of the insulin receptor, which suggests that insulin may influence GβL expression. Indeed, GβL protein and mRNA levels, in fully differentiated 3T3-L1 adipocytes, were upregulated by insulin in a concentration-dependent fashion. These changes were highly sensitive to insulin stimulation, being minimally affected by doses as low as 0.1 nM and maximally elevated by 1 nM doses. These data suggest that insulin regulates GβL production and imply that some of the actions of insulin may be mediated, in part, by this novel intracellular protein.