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(1b)-adrenergic receptor is postul ated to be initiated by disruption of an interhelical salt-bridge constrain t between an aspartic acid (Asp-125) and a lysine residue (Lys-331) in tran smembrane domains three and seven, respectively. Single point mutations tha t disrupt the charges of either of these residues results in constitutive a ctivity. To validate this hypothesis, we used site-directed mutagenesis to switch the position of these amino acids to observe, if possible, regenerat ion of the salt-bridge reverses that the constitutive activity of the singl e point mutations. The transiently expressed switch mutant receptor display ed an altered pharmacological profile, The affinity of selective Agonist-de pendent activation of the alpha(1)-adrenergic receptor is postulated to be initiated by disruption of an interhelical salt-bridge constraint between a n aspartic acid (Asp-125) and a lysine residue (Lys-331) in transmembrane d omains three and seven, respectively. Single point mutations that disrupt t he 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 mut ations. The transiently expressed switch mutant receptor displayed an alter ed pharmacological profile, The affinity of selective alpha(1b)-adrenergic receptor antagonists for the switch mutant (D125K/K331D) was no different f rom the wild-type alpha(1b)-adrenergic receptor, suggesting that both recep tors are maintaining similar tertiary structures in the cell membrane. Howe ver, there was a significant 4-6-fold decrease in the affinity of protonate d amine receptor agonists and a 3-6-fold increase in the affinity of carbox ylated catechol derivatives for the switch mutant compared with the wild-ty pe alpha(1b)-adrenergic receptor. This pharmacology is consistent with a re versed charge at position 125 in transmembrane domain three. Interestingly, the ability of either a negatively or positively charged agonist to genera te soluble inositol phosphates was similar for both types of receptors, Fin ally, the switch mutant (D125K/K331D) displayed similar basal signaling act ivity as the wild-type receptor, reversing the constitutive activity of the single point mutations (D125K and K331D), This suggests an ionic constrain t has been reformed in the snitch mutant analogous to the restraint previou sly described for the wild-type alpha(1b)-adrenergic receptor. These result s strongly establish the disruption of an electrostatic interaction as an i nitial step in the agonist-dependent activation of alpha(1)-adrenergic rece ptors.