The N54-Alpha(S) As Mutant Has Decreased Affinity For Beta Gamma And Suggests A Mechanism For Coupling Heterotrimeric G Protein Nucleotide Exchange With Subunit Dissociation

Ser54 of G(s)alpha 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 alpha(i/o), this results from persistent binding of alpha to beta gamma, whereas for small GTP binding proteins and alpha(s) this results from persistent binding to guanine nucleotide exchange factor or receptor. This work examined the role of beta gamma interactions in mediating the properties of the Ser54-like mutants of G alpha subunits. Unexpectedly, WT-alpha(s) or N54-alpha(s) coexpressed with alpha(1B)-adrenergic receptor in human embryonic kidney 293 cells decreased receptor stimulation of IP3 production by a cAMP-independent mechanism, but WT-alpha(s) was more effective than the mutant. One explanation for this result would be that alpha(s), like Ser47 alpha(i/o), blocks receptor activation by sequestering beta gamma; implying that N54-alpha(S) has reduced affinity for beta gamma since it was less effective at blocking IP3 production. This possibility was more directly supported by the observation that WT-alpha(s) was more effective than the mutant in inhibiting beta gamma activation of phospholipase C beta 2. Further, in vitro synthesized N54-alpha(s) bound biotinylated-beta gamma with lower apparent affinity than did WT-alpha(s). The Cys54 mutation also decreased beta gamma binding but less effectively than N54-alpha(s). Substitution of the conserved Ser in alpha(o) with Cys or Asn increased beta gamma binding, with the Cys mutant being more effective. This suggests that Ser54 of alpha(s) is involved in coupling changes in nucleotide binding with altered subunit interactions, and has important implications for how receptors activate G proteins.