ACTIVATION OF THE ALPHA(1B)-ADRENERGIC RECEPTOR IS INITIATED BY DISRUPTION OF AN INTERHELICAL SALT BRIDGE CONSTRAINT

Rhodopsin receptor activation in involves the disruption of a salt bri dge constraint between glutamic acid 113 on transmembrane 3 and a lysi ne 296 in transmembrane 7, which forms a Schiffs base with retinal, Li ght-induced isomerization of cis-retinal to the all trans form breaks this rhodopsin salt bridge leading to receptor activation, The analogo us residues in alpha(1b)-adrenergic receptors, aspartic acid 125 and l ysine 331, also have the potential of forming a constraining salt brid ge holding the receptor to an inactive protein configuration, This alp ha(1b)-adrenergic receptor salt bridge constraint is then released upo n binding by the receptor agonist. To test this hypothesis, site-direc ted mutagenesis was used to eliminate the positive charge at position 331 by substitution of an alanine, The expressed alpha(1b)-adrenergic receptor mutant demonstrated a 6-fold increased epinephrine binding af finity with no alterations of affinity values for selective adrenergic receptor antagonists, Furthermore, an increased epinephrine potency f or total soluble inositol phosphate production along with an elevated basal inositol triphosphate level was observed in COS-1 cells transfec ted with mutant versus wild-type alpha(1b)-adrenergic receptors, Simil ar results were obtained for a lysine to a glutamic acid alpha(1b)-adr energic receptor mutation. In addition, increased basal inositol triph osphate levels were also observed for two aspartic acid 125 alpha(1b)- adrenergic receptor mutations, consistent with this residue's role as the counterion of the salt bridge, Taken together, these alpha(1b)-adr energic receptor mutations suggest a molecular mechanism by which the positively charged lysine 331 stabilizes the negatively charged aspart ic acid 125 via a salt bridge constraint until bound by the receptor a gonist.