IDENTIFICATION OF A CONSERVED SWITCH RESIDUE RESPONSIBLE FOR SELECTIVE CONSTITUTIVE ACTIVATION OF THE BETA(2)-ADRENERGIC RECEPTOR

A cysteine-to-phenylalanine mutation of residue 116 in the third trans membrane domain of the beta(2)-adrenergic receptor caused selective co nstitutive activation of Na+/H+ exchange through a pathway not involvi ng cAMP. This selectivity was identified by comparing binding and sign aling characteristics of wild-type (WT) versus C116F mutant receptors transiently transfected into COS-1 cells, Indicating constitutive acti vity, 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 productio n was not exhibited by the C116F mutant, a constitutive stimulation of the Na+/H+ exchanger (NHE1) was observed. This was identified by meas uring either basal intracellular pH (pH(i)) or rate of pH(i) recovery from cellular acid load. Due to a higher rate of H+ efflux through NHE 1, C116F transfectants exhibited a significantly higher pH(i) (7.42) t han did WT transfectants (7.1). Furthermore, the rate of pH(i) recover y from acid load facilitated by NHE1 was 8.1-fold faster in mutant tra nsfectants than in WT transfectants. The lower rate seen in the WT cas e was stimulated by epinephrine, and the higher rate seen in the mutan t case was inhibited by ICI 118551. These findings, which show that a C116F mutation of the beta(2)-adrenergic receptor evokes selective con stitutive coupling to NHE1 over cAMP, form the basis of our prediction that multiple and distinct activation states can exist in G protein-c oupled receptors.