A novel mutation in the switch 3 region of Gsα in a patient with Albright hereditary osteodystrophy impairs GDP binding and receptor activation

Albright hereditary osteodystrophy (AHO), a disorder characterized by skeletal abnormalities and obesity, is associated with heterozygous inactivating mutations in the gene for G(s)alpha. A novel G(s)alpha mutation encoding the substitution of tryptophan for a nonconserved arginine within the switch 3 region (G(s)alpha R258W) was identified in an AHO patient. Although reverse transcription-polymerase chain reaction studies demonstrated that mRNA expression from wild type and mutant alleles was similar, G(s)alpha expression in erythrocyte membranes from the affected patient was reduced by 50%. A G(s)alpha R258W cDNA, as well as one with arginine replaced by alanine (G(s)alpha R258A), was generated, and the biochemical properties of lit vitro transcription/translation products were examined, When reconstituted with cyc(-) membranes, both mutant proteins were able to stimulate adenylyl cyclase normally in the presence of guanosine-5'-O-(3-thiotriphosphate) (GTP gamma S) but had decreased ability in the presence of isoproterenol or AlF4- (a mixture of 10 mu M AlCl3 and 10 mM NaF). The ability of each mutant to bind and be activated by GTP gamma S or AlF4- was assessed by trypsin protection assays. Both mutants were protected normally by GTP gamma S but showed reduced protection in the presence of AlF4-. The addition of excess GDP (2 mM) was able to rescue the ability of AlF4- to protect the mutants, suggesting that they might have reduced affinity for GDP. A G(s)alpha R258A mutant purified from Escherichia coli had decreased affinity for GDP and an apparent rate of GDP release that was 10-fold greater than that of wild type G(s)alpha. Sucrose density gradient analysis demonstrated that both G(s)alpha R258W and G(s)alpha R258A were thermolabile at higher temperatures and that denaturation of both mutants was prevented by the presence of 0.1 mM GTP gamma S or 2 mM GDP. The crystal structure of G(s)alpha demonstrates that Arg(258) interacts with a conserved residue in the helical domain (Gln(170)). Arg(258) substitutions would be predicted to open the cleft between the GTPase and helical domains, allowing for increased GDP release in the inactive state, resulting in enhanced thermolability and reduced AlF4--induced adenylyl cyclase stimulation and trypsin protection, since activation by AlF4- requires bound GDP.