Contribution of serine residues to constitutive and agonist- induced signaling via the D(2S) dopamine receptor

Evidence for multiple, agonist- specific active conformations

Brenda L. Wiens, Cole S. Nelson, Kim Neve

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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-toalanine 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(2S) 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.

Original languageEnglish (US)
Pages (from-to)435-444
Number of pages10
JournalMolecular Pharmacology
Volume54
Issue number2
StatePublished - Aug 1998

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Dopamine Receptors
Serine
Guanosine 5'-O-(3-Thiotriphosphate)
Dopamine
Mutation
Dopamine D2 Receptors
Xenopus laevis
Guanosine Triphosphate
GTP-Binding Proteins
Adenylyl Cyclases
Glioma
Oocytes
dihydrexidine

ASJC Scopus subject areas

  • Pharmacology

Cite this

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title = "Contribution of serine residues to constitutive and agonist- induced signaling via the D(2S) dopamine 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 D(2S) dopamine receptor mutants, each containing a serine-toalanine 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(2S) 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.",
author = "Wiens, {Brenda L.} and Nelson, {Cole S.} and Kim Neve",
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T1 - Contribution of serine residues to constitutive and agonist- induced signaling via the D(2S) dopamine receptor

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AU - Nelson, Cole S.

AU - Neve, Kim

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N2 - 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-toalanine 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(2S) 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.

AB - 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-toalanine 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(2S) 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.

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