The N54-α_s Mutant Has Decreased Affinity for βγ and Suggests a Mechanism for Coupling Heterotrimeric G Protein...

Ser54 of G_sα binds guanine nucleotide and Mg²⁺ as part of a conserved sequence motif in GTP binding proteins. Mutating the homologous residue in small and heterotrimeric G proteins generates dominant-negative proteins, but by protein-specific mechanisms. For α_i/o, this results from persistent binding of α to βγ, whereas for small GTP binding proteins and α_s this results from persistent binding to guanine nucleotide exchange factor or receptor. This work examined the role of βγ interactions in mediating the properties of the Ser54-like mutants of Gα subunits. Unexpectedly, WT-α_s or N54-α_s coexpressed with α_1B-adrenergic receptor in human embryonic kidney 293 cells decreased receptor stimulation of IP3 production by a cAMP-independent mechanism, but WT-α_s was more effective than the mutant. One explanation for this result would be that α_s, like Ser47 α_i/o, blocks receptor activation by sequestering βγ; implying that N54-α_S has reduced affinity for βγ since it was less effective at blocking IP3 production. This possibility was more directly supported by the observation that WT-α_s was more effective than the mutant in inhibiting βγ activation of phospholipase Cβ2. Further, in vitro synthesized N54-α_s bound biotinylated-βγ with lower apparent affinity than did WT-α_s The Cys54 mutation also decreased βγ binding but less effectively than N54-α_s Substitution of the conserved Ser in α_o with Cys or Asn increased βγ binding, with the Cys mutant being more effective. This suggests that Ser54 of α_s is involved in coupling changes in nucleotide binding with altered subunit interactions, and has important implications for how receptors activate G proteins.