Contribution of Serine Residues to Constitutive and Agonist-Induced Signaling via the D_2SDopamine Receptor: Evidence for Multiple, Agonist-Specific Active Conformations

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 D2S 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 approximately 50%. Finally, constitutive stimulation of [35S]guanosine-5'-O-(3-thio)triphosphate binding and inhibition of adenylate cyclase by the D2S receptor 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.

The Forgotten Serine

“…This is consistent with experimental results in the D 2 receptor implicating each of the three serines in the binding of various ligands, although different serines were found to be more or less critical with different ligands (11,12). More recently we have applied the substituted cysteine accessibility method to the aligned portion of TM5 in the human ␤ 2 AR, 2 and we were surprised to observe that substitution of Ser-203 5.42 by Cys, unlike the published mutation of this Ser to Ala in the hamster ␤ 2 AR (4), did not impair expression of the receptor but instead lowered its affinity for both isoproterenol and for the radiolabeled antagonist CGP-12177.…”

A QM protein–ligand investigation of antipsychotic drugs with the dopamine D2 Receptor (D2R)

“…The increased H-bond distance is consistent with the decreased binding energies. All these energy analyses revealed a satisfactory agreement with the experimental results (Fu et al, 1996;Javitch, Ballesteros, Weinstein, & Chen, 1998;Wiens, Nelson, & Neve, 1998). However, it should be noted that such analyses can be very sensitive to the dynamics and motion of the compounds in the ligand binding pocket.…”

Estrogen inhibits D2S receptor-regulated Gi3 and Gs protein interactions to stimulate prolactin production and cell proliferation in lactotropic cells

“…As described by an extended allosteric ternary complex model of G protein-coupled receptor activation, receptors spontaneously isomerize between active and inactive conformations, so receptors can modulate signaling pathways in the absence of an agonist (Strange 1999). In addition, significant constitutive activity of recombinant D2S receptors expressed in mammalian cells has been described previously (Wiens et al 1998). Hence, the ligand-independent changes in hormone production and cell proliferation were due to constitutively activated D2 receptors in transfected cells.…”