Mutation in a protein kinase C phosphorylation site of the 5-HT1A receptor preferentially attenuates Ca2+ responses to partial as opposed to higher-efficacy 5-HT1A agonists.
The Thr(149)Ala mutation in a putative protein kinase C phosphorylation site of the 5-HT(1A) receptor's second intracellular loop has been shown to affect the closing of Ca(2+) channels and Ca(2+) mobilisation without interfering with the inhibitory cAMP pathway (Mol Pharmacol 52 (1997) 164). Here, the Ca(2+) responses for a series of 5-HT(1A) agonists were compared between the wild-type (wt) and mutant Thr(149)Ala 5-HT(1A) receptor as part of a fusion protein containing a G(alpha)(15) protein. Neither the mutation nor the fusion process modified the [(3)H]WAY 100635-based ligand binding profile of the fusion proteins as compared to the wt 5-HT(1A) receptor protein. Whereas at the wt 5-HT(1A) receptor, 5-HT induced a Ca(2+) response in CHO-K1 cells via endogenous G(i/o) proteins, the Ca(2+) response to 5-HT at the mutant Thr(149)Ala 5-HT(1A) receptor was fully dependent on either the co-expression or the fusion to a recombinant G(alpha)(15) protein. Buspirone, flesinoxan and 8-OH-DPAT produced a graded partial response (26 to 62%) at the wt 5-HT(1A):G(alpha)(15) fusion protein; F 13640, 5-CT and F 14679 behaved as higher-efficacy agonists with maximal Ca(2+) responses similar to 5-HT. The maximal Ca(2+) responses at the mutant Thr(149)Ala 5-HT(1A):G(alpha)(15) fusion protein were significantly attenuated for flesinoxan and 8-OH-DPAT (-45 and -36%, respectively); the response to the other 5-HT agonists was not significantly affected. A similar effect was observed upon treatment with phorbol 12-myristate 13-acetate at the Thr(149)Ala 5-HT(1A):G(alpha)(15) fusion protein. In conclusion, the amplitude of the Ca(2+) responses induced by partial, but not that to fuller 5-HT(1A) receptor agonists, is affected by the Thr(149)Ala mutation of the 5-HT(1A):G(alpha)(15) fusion protein.
Wurch
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T
Department of Cellular and Molecular Biology, Centre de Recherche Pierre Fabre, 17, avenue Jean Moulin, 81106 Castres Cédex, France.
Colpaert
F C
FC
Pauwels
P J
PJ
D017371:8-Hydroxy-2-(di-n-propylamino)tetralin / Q000494:pharmacology; D000818:Animals; D016466:CHO Cells; D002118:Calcium / Q000502:physiology*; D006224:Cricetinae; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D020962:Heterotrimeric GTP-Binding Proteins / Q000235:genetics; D006801:Humans; D008024:Ligands; D009154:Mutation / Q000235:genetics* / Q000502:physiology*; D010766:Phosphorylation; D010879:Piperazines / Q000494:pharmacology; D011493:Protein Kinase C / Q000235:genetics*; D011725:Pyridines / Q000494:pharmacology; D011869:Radioligand Assay; D011985:Receptors, Serotonin / Q000187:drug effects / Q000235:genetics*; D044263:Receptors, Serotonin, 5-HT1; D012702:Serotonin Antagonists / Q000494:pharmacology; D017366:Serotonin Receptor Agonists / Q000494:pharmacology*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014162:Transfection
D008024:Ligands; D010879:Piperazines; D011725:Pyridines; D011985:Receptors, Serotonin; D044263:Receptors, Serotonin, 5-HT1; D012702:Serotonin Antagonists; D017366:Serotonin Receptor Agonists; C056895:flesinoxan; C090413:N-(2-(4-(2-methoxyphenyl)-1-piperazinyl)ethyl)-N-(2-pyridinyl)cyclohexanecarboxamide; D017371:8-Hydroxy-2-(di-n-propylamino)tetralin; D011493:Protein Kinase C; C112631:G protein alpha 16; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D020962:Heterotrimeric GTP-Binding Proteins; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium