Abstract
Ser54 of Gsα 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 αi/o this results from persistent binding of α to β γ, whereas for small GTP binding proteins and αs, 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, wild type (wt) or N54-αs co-expressed with α 1B-adrenergic receptor in HEK293 cells decreased receptor stimulation of IP3 production by a cAMP-independent mechanism; but wt-αs was more effective than the mutant. One explanation for this was that αs, like Ser47 αi/o mutants, blocks receptor activation by sequestering βγ; implying that N54-αs 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 PLCβ2. Further, in vitro synthesized N54-αs bound biotinylated-βγ with lower apparent affinity than did wt-αs. The Cys54 mutation also decreased βγ binding but less effectively than N54-αs. Substitution of the conserved Ser in αo with Cys or Asn increased βγ binding, with the Cys mutant being more effective. This suggest that Ser54 of αs is involved in coupling changes in nucleotide binding with altered subunit interactions, and has important implications for how receptors activate G proteins.
- alpha-adrenergic receptors
- G protein coupled signaling
- g protein-coupled receptors (GPCRS)
- signal transduction
- The American Society for Pharmacology and Experimental Therapeutics