IDENTIFICATION OF AMINO-ACID-RESIDUES OF GS-ALPHA CRITICAL TO REPRESSION OF ADIPOGENESIS

Gs alpha regulates the differentiation of 3T3-L1 mouse embryonic fibro blasts to adipocytes, a process termed adipogenesis, Through the expre ssion of chimera created by substituting regions of Gs alpha with corr esponding regions of the G protein Gi alpha 2, the domain of Gs alpha involved in repression of adipogenesis was localized to sequence 146-2 35 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-scan ning mutagenesis, chimeras in Gs alpha constructed from trisection of the sequence 125-213 of Gi alpha 2 mere expressed stably, and clones w ere evaluated for the ability of the chimera to repress adipogenesis i n response to the inducers, dexamethasone and methylisobutylxanthine, in combination. The chimera containing sequence 150-177 of Gi alpha 2 repressed adipogenesis, whereas the chimeras with either sequence 125- 149 or 178-213 of Gi alpha 2 failed to repress induction of adipogenes is, Alanine-scanning mutagenesis of these two critical domains was per formed first in clusters and then confirmed by analysis of single muta tions. Six residues unique to Gs alpha were identified as critical to repression of adipogenesis, Asn(167), Cys(200), Leu(203), Ser(205), Va l(214), and Lys(216). Leu(208) and Ser(205) are required in tandem, as mutagenesis to alanine of either one alone was without effect on repr essor activity. The remaining four residues are required for repressor activity; mutation of any one of these abolishes the ability of Gsa t o repress adipogenesis, although not affecting the ability of the muta nt form of Gs alpha to regulate adenylylcyclase, Using conserved landm arks found in the crystal structures of Gi alpha and Gs alpha, the Leu (203) and Ser(205) cluster appears to be exposed, closely aligned and located in switch I region. Asn(167), Val(214), and Lys(216) project t o regions on Gs alpha that are exposed in the GTP gamma S-liganded sta te of the cu subunit. We speculate that these residues constitute an i mportant contact domain between Gs alpha and the effector controlling adipogenesis, which is yet to be identified.