Characteristics for a salt-bridge switch mutation of the alpha(1b) adrenergic receptor. Altered pharmacology and rescue of constitutive activity.
Agonist-dependent activation of the alpha(1)-adrenergic receptor is postulated to be initiated by disruption of an interhelical salt-bridge constraint between an aspartic acid (Asp-125) and a lysine residue (Lys-331) in transmembrane domains three and seven, respectively. Single point mutations that disrupt the charges of either of these residues results in constitutive activity. To validate this hypothesis, we used site-directed mutagenesis to switch the position of these amino acids to observe, if possible, regeneration of the salt-bridge reverses that the constitutive activity of the single point mutations. The transiently expressed switch mutant receptor displayed an altered pharmacological profile. The affinity of selective alpha(1b)-adrenergic receptor antagonists for the switch mutant (D125K/K331D) was no different from the wild-type alpha(1b)-adrenergic receptor, suggesting that both receptors are maintaining similar tertiary structures in the cell membrane. However, there was a significant 4-6-fold decrease in the affinity of protonated amine receptor agonists and a 3-6-fold increase in the affinity of carboxylated catechol derivatives for the switch mutant compared with the wild-type alpha(1b)-adrenergic receptor. This pharmacology is consistent with a reversed charge at position 125 in transmembrane domain three. Interestingly, the ability of either a negatively or positively charged agonist to generate soluble inositol phosphates was similar for both types of receptors. Finally, the switch mutant (D125K/K331D) displayed similar basal signaling activity as the wild-type receptor, reversing the constitutive activity of the single point mutations (D125K and K331D). This suggests an ionic constraint has been reformed in the switch mutant analogous to the restraint previously described for the wild-type alpha(1b)-adrenergic receptor. These results strongly establish the disruption of an electrostatic interaction as an initial step in the agonist-dependent activation of alpha(1)-adrenergic receptors.
Porter
J E
JE
Department of Molecular Cardiology, Lerner Research Institute, The Cleveland Clinic Foundation, Cleveland, Ohio 44195, USA.
Perez
D M
DM
D058646:Adrenergic alpha-1 Receptor Agonists; D058668:Adrenergic alpha-1 Receptor Antagonists; D000596:Amino Acids / Q000378:metabolism; D000818:Animals; D019556:COS Cells; D002395:Catecholamines / Q000493:pharmacokinetics; D007295:Inositol Phosphates / Q000378:metabolism; D007477:Ions; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010656:Phenylephrine / Q000493:pharmacokinetics; D011485:Protein Binding; D018340:Receptors, Adrenergic, alpha-1 / Q000235:genetics* / Q000378:metabolism*
D058646:Adrenergic alpha-1 Receptor Agonists; D058668:Adrenergic alpha-1 Receptor Antagonists; D000596:Amino Acids; D002395:Catecholamines; D007295:Inositol Phosphates; D007477:Ions; D018340:Receptors, Adrenergic, alpha-1; D010656:Phenylephrine