Identification of amino acid residues of Gsα critical to repression of adipogenesis

Gsα 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 Gsα with corresponding regions of the G protein Giα2, the domain of Gsα 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 Gsα constructed from trisection of the sequence 125-213 of Giα2 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 Giα2 repressed adipogenesis, whereas the chimeras with either sequence 125-149 or 178-213 of Giα2 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 Gsα were identified as critical to repression of adipogenesis, Asn¹⁶⁷, Cys²⁰⁰, Leu²⁰³, Set²⁰⁵, 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 Gsα to repress adipogenesis, although not affecting the ability of the mutant form of Gsα to regulate adenylylcyclase. Using conserved landmarks found in the crystal structures of Giα1 and Gsα, the Leu²⁰³ and Ser²⁰⁵ cluster appears to be exposed, closely aligned and located in switch I region. Ash¹⁶⁷, Val²¹⁴, and Lys²¹⁶ project to regions on Gsα that are exposed in the GTPγS-liganded state of the α subunit. We speculate that these residues constitute an important contact domain between Gsα and the effector controlling adipogenesis, which is yet to be identified.