Identification of a conserved switch residue responsible for selective constitutive activation of the...

A cysteine-to-phenylalanine mutation of residue 116 in the third transmembrane domain of the beta2-adrenergic receptor caused selective constitutive activation of Na+/H+ exchange through a pathway not involving cAMP. This selectivity was identified by comparing binding and signaling characteristics of wild-type (WT) versus C116F mutant receptors transiently transfected into COS-1 cells. Indicating constitutive activity, ligand binding to the C116F mutant showed a 78-fold higher than WT affinity for isoproterenol and a 40-fold lower than WT affinity for ICI 118551. Although agonist-independent activation of cAMP production was not exhibited by the C116F mutant, a constitutive stimulation of the Na+/H+ exchanger (NHE1) was observed. This was identified by measuring either basal intracellular pH (pHi) or rate of pHi recovery from cellular acid load. Due to a higher rate of H+ efflux through NHE1, C116F transfectants exhibited a significantly higher pHi (7.42) than did WT transfectants (7.1). Furthermore, the rate of pHi recovery from acid load facilitated by NHE1 was 2.1-fold faster in mutant transfectants than in WT transfectants. The lower rate seen in the WT case was stimulated by epinephrine, and the higher rate seen in the mutant case was inhibited by ICI 118551. These findings, which show that a C116F mutation of the beta2-adrenergic receptor evokes selective constitutive coupling to NHE1 over cAMP, form the basis of our prediction that multiple and distinct activation states can exist in G protein-coupled receptors.