Abstract
Productive interaction between receptors and G proteins involves multiple intracellular receptor domains, but the role of individual receptor amino acids in directing the selection of specific signaling pathways has not yet been identified. Sequence alignment of several G protein-coupled receptors identified a highly conserved threonine residue in the i2 loop of the 5-hydroxytryptamine 1A (5-HT1A) receptor that is a putative protein kinase C phosphorylation consensus site and is located in a predicted amphipathic α-helical domain. To examine the role of this conserved threonine residue in 5-HT1A receptor coupling to Gi/Go proteins, this residue was mutated to alanine (T149A mutant). Wild-type and mutant 5-HT1Areceptors were stably transfected into both Ltk− and GH4C1 cells to investigate receptor coupling to multiple signaling pathways. In both cell lines, the T149A mutant displayed similar agonist affinities as the wild-type receptor. In Ltk− cells, the T149A 5-HT1A receptor inhibited cAMP accumulation by 30% compared with wild-type (83%). A 2.6-fold increase in intracellular calcium (due to phospholipase C-mediated calcium mobilization) was observed for the wild-type receptor upon the addition of 100 nm 5-HT; whereas the T149A 5-HT1A receptor failed to mediate a calcium mobilization response at equivalent receptor levels to wild-type. When transfected in GH4C1 cells, the T149A receptor mutant fully inhibited basal cAMP and partially inhibited Gs-stimulated cAMP accumulation compared with wild-type receptor (57 ± 14% versus 86 ± 2%). In contrast, the T149A 5-HT1A receptor mutant failed to block the influx of calcium induced by calcium channel agonist (±)-Bay K8644, whereas the wild-type 5-HT1A receptor inhibited the calcium influx by 40%. Thus, the Thr149 residue is directly involved in G protein coupling to calcium mobilization (mediated by βγ subunits of Gi2) and to inhibition of calcium channel activation (mediated by βγ subunits of Go) but plays a minor role in coupling to αi-mediated inhibition of cAMP accumulation. The conserved i2 loop threonine may serve as a G protein contact site to direct the signaling specificity of multiple receptors.
Footnotes
- Received January 21, 1997.
- Accepted April 9, 1997.
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Send reprint requests to: Dr. Paul R. Albert, Neuroscience Research Institute, University of Ottawa, 451 Smyth Road, Ottawa, Canada K1H 8M5. E-mail: palbert{at}uottawa.ca
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This work was supported by the Medical Research Council (MRC), Canada. P.R.A. is Ciba Geigy/MRC Michael Smith Professor.
- The American Society for Pharmacology and Experimental Therapeutics
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