Identification of amino acid residues of Gsalpha critical to repression of adipogenesis.

Gsalpha regulates the differentiation of 3T3-L1 mouse embryonic fibroblasts to adipocytes, a process termed adipogenesis. Through the expression of chimera created by substituting regions of Gsalpha with corresponding regions of the G protein Gialpha2, the domain of Gsalpha involved in repression of adipogenesis was localized to sequence 146-235 of the molecule (Wang, H-y., Johnson, G. L., Liu, X. , Malbon, C. C. (1996) J. Biol. Chem. 271, 22022-22029). As a prelude to alanine-scanning mutagenesis, chimeras in Gsalpha constructed from trisection of the sequence 125-213 of Gialpha2 were expressed stably, and clones were evaluated for the ability of the chimera to repress adipogenesis in response to the inducers, dexamethasone and methylisobutylxanthine, in combination. The chimera containing sequence 150-177 of Gialpha2 repressed adipogenesis, whereas the chimeras with either sequence 125-149 or 178-213 of Gialpha2 failed to repress induction of adipogenesis. Alanine-scanning mutagenesis of these two critical domains was performed first in clusters and then confirmed by analysis of single mutations. Six residues unique to Gsalpha were identified as critical to repression of adipogenesis, Asn167, Cys200, Leu203, Ser205, Val214, and Lys216. Leu203 and Ser205 are required in tandem, as mutagenesis to alanine of either one alone was without effect on repressor activity. The remaining four residues are required for repressor activity; mutation of any one of these abolishes the ability of Gsalpha to repress adipogenesis, although not affecting the ability of the mutant form of Gsalpha to regulate adenylylcyclase. Using conserved landmarks found in the crystal structures of Gialpha1 and Gsalpha, the Leu203 and Ser205 cluster appears to be exposed, closely aligned and located in switch I region. Asn167, Val214, and Lys216 project to regions on Gsalpha that are exposed in the GTPgammaS-liganded state of the alpha subunit. We speculate that these residues constitute an important contact domain between Gsalpha and the effector controlling adipogenesis, which is yet to be identified.