Abstract

Dopamine D₂ receptors contain a cluster of serine residues in the fifth transmembrane domain that contribute to activation of the receptor as well as to the binding of agonists. We used rat D_2S dopamine receptor mutants, each containing a serine-to-alanine substitution (S193A, S194A, S197A), to investigate the mechanism through which these residues affect activation of the receptor. Activation of the mutant receptor S194A was abolished in an agonist-dependent manner, such that dopamine no longer inhibited cAMP accumulation in C6 glioma cells or activated G protein-regulated K⁺ channels in Xenopus laevis oocytes, whereas the efficacy of several other agonists was unaffected. Dihydrexidine did not inhibit cAMP accumulation at either S193A or S194A. The decreased efficacy of dihydrexidine at S193A and S194A and dopamine at S194A was associated with a decreased ability to detect a GTP-sensitive high affinity binding state for these agonists. The ability of dopamine to stimulate [³⁵S]guanosine-5′-O-(3-thio)triphosphate binding via S194A also was decreased by ∼50%. Finally, constitutive stimulation of [³⁵S]guanosine-5′-O-(3-thio)triphosphate binding and inhibition of adenylate cyclase by the D_2Sreceptor was reduced by mutation of either S193 or S194. These data support the existence of multiple active receptor conformations that are differentially sensitive to mutation of serine residues in the fifth-transmembrane domain.