Physicochemical Modulation of Agonist-Induced [35S]GTPγS Binding: Implications for Coexistence of Multiple Functional Conformations of Dopamine D1-Like Receptors

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Abstract
Dopamine agonist-stimulated [35S]GTPgammaS binding to membrane G proteins was studied in select brain regions under experimental conditions that permit the activation of receptor coupling to the G proteins Gi, Gs, or Gq. Agents studied were agonists known to be effective at various dopamine receptor/effector systems and included quinelorane (D2-like/Gi), SKF38393 (D1-like/Gq, D1-like/Gs), SKF85174 (D1-like/Gs), and SKF83959 (D1-like/Gq). Dopamine and SKF38393 significantly stimulated [35S]GTPgammaS binding to normal striatal membranes by 161% and 67% above controls. Deoxycholate, which enhances agonist-induced phospholipase C (PLC) stimulation, markedly enhanced the agonistic effects of dopamine and SKF38393 to 530% and 637% above controls, respectively. The enhancing effects of deoxycholate were reversed if it was washed off the membranes before agonist addition. The thiol-reducing agent, dithiothreitol, completely abolished the effects of SKF38393 and SKF83959, whereas SKF85174 effects were augmented. Agonist responses were concentration-related, and highest efficacies were obtained in the hippocampus, thus paralleling both the brain regional distribution and agonist efficacies previously observed in phosphoinositide hydrolysis assays. These findings suggest that D1-like receptor conformations that mediate agonist stimulation of Gs/adenylylcyclase may be structurally different from those that mediate Gq/PLC activation. Although the exact mechanism of deoxycholate's effect awaits elucidation, the results are consistent with the emerging concept of functional selectivity whereby deoxycholate could create a membrane environment that facilitates the transformation of the receptor from a conformation that activates Gs/adenylylcyclase to one that favors Gq/PLC signaling.