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 (Gbeta). Although chemically and structurally similar to Gbeta subunits, the predicted amino acid sequence, when compared with the previously cloned Gbeta isoforms, was found to be only 31-41% similar and thus was named Gbeta-like (GbetaL, '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 GbetaL 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 GbetaL 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 GbetaL. The ubiquitous nature of the tissue expression pattern of GbetaL is similar to that of the insulin receptor, which suggests that insulin may influence GbetaL expression. Indeed, GbetaL 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 GbetaL production and imply that some of the actions of insulin may be mediated, in part, by this novel intracellular protein.
Rodgers
B D
BD
Diabetes Section, Gerontology Research Center, National Institute on Aging, National Institutes of Health, 5600 Nathan Shock Drive, Baltimore, Maryland 21224, USA. drodgers@jhmi.edu
Levine
M A
MA
Bernier
M
M
Montrose-Rafizadeh
C
C
D017667:Adipocytes / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D018076:DNA, Complementary / Q000235:genetics; D004305:Dose-Response Relationship, Drug; D015870:Gene Expression; D015723:Gene Library; D020962:Heterotrimeric GTP-Binding Proteins / Q000096:biosynthesis / Q000235:genetics; D007328:Insulin / Q000494:pharmacology / Q000502:physiology*; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014018:Tissue Distribution; D015854:Up-Regulation / Q000187:drug effects
D018076:DNA, Complementary; D007328:Insulin; D012333:RNA, Messenger; D020962:Heterotrimeric GTP-Binding Proteins