Intragenic suppressor mutations of a constitutively active allele of Gs alpha

Abstract

Heterotrimeric G‐proteins cycle between an active/GTP‐bound conformation and an inactive/GDP‐bound conformation. Reduction in GTP hydrolysis by the G‐protein alpha subunit results in constitutive activity of the protein. McCune‐Albright Syndrome (MAS) is a human genetic disorder caused by a mutation that constitutively activates the Gs alpha subunit by inhibiting GTP hydrolysis. Arginine 201 substitutions with histidine, serine, or cysteine have been reported in biopsies from MAS patients. Previous work in this laboratory modeled the MAS mutation in a yeast system, and identified an intragenic suppressor of the MAS mutation, which substituted two residues in the GTP‐binding site. The homologous mutations (F222P, D223V) in the human Gs alpha subunit also suppressed the constitutive activity of the MAS mutation when the protein was expressed in human cells. To extend these studies, F222 and D223 were mutated to a variety of other amino acids to determine the structural requirements for suppression of the MAS mutation. Basal and receptor‐stimulated cAMP levels were measured in HEK293 cells transfected with the different alleles of Gs alpha. While some single mutations at either F222 or D223 were able to suppress the MAS R201H mutation, a range of levels of suppression were observed.