The N54- Mutant Has Decreased Affinity for and Suggests a Mechanism for Coupling Heterotrimeric G Protein Nucleotide Exchange with Subunit Dissociation.

Ser54 of G binds guanine nucleotide and Mg 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 , this results from persistent binding of to , whereas for small GTP binding proteins and this results from persistent binding to guanine nucleotide exchange factor or receptor. This work examined the role of interactions in mediating the properties of the Ser54-like mutants of G subunits. Unexpectedly, WT- or N54- coexpressed with -adrenergic receptor in human embryonic kidney 293 cells decreased receptor stimulation of IP3 production by a cAMP-independent mechanism, but WT- was more effective than the mutant. One explanation for this result would be that , like Ser47 , blocks receptor activation by sequestering ; implying that N54- has reduced affinity for since it was less effective at blocking IP3 production. This possibility was more directly supported by the observation that WT- was more effective than the mutant in inhibiting activation of phospholipase C2. Further, in vitro synthesized N54- bound biotinylated- with lower apparent affinity than did WT- The Cys54 mutation also decreased binding but less effectively than N54- Substitution of the conserved Ser in with Cys or Asn increased binding, with the Cys mutant being more effective. This suggests that Ser54 of is involved in coupling changes in nucleotide binding with altered subunit interactions, and has important implications for how receptors activate G proteins.