A CONSERVED THREONINE RESIDUE IN THE 2ND INTRACELLULAR LOOP OF THE 5-HYDROXYTRYPTAMINE 1A RECEPTOR DIRECTS SIGNALING SPECIFICITY

Productive interaction between receptors and G proteins involves multi ple intracellular receptor domains, but the role of individual recepto r amino acids in directing the selection of specific signaling pathway s 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 locate d in a predicted amphipathic alpha-helical domain. To examine the role of this conserved threonine residue in 5-HT1A receptor coupling to G( i)/G(o) proteins, this residue was mutated to alanine (T149A mutant). Wild-type and mutant 5-HT1A receptors were stably transfected into bot h Ltk(-) and GH4C1 cells to investigate receptor coupling to multiple signaling pathways. In both cell lines, the T149A mutant displayed sim ilar agonist affinities as the wild-type receptor. In Ltk(-) cells, th e T149A 5-HT1A receptor inhibited cAMP accumulation by 30% compared wi th 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 T149 A 5-HT1A receptor failed to mediate a calcium mobilization response at equivalent receptor levels to wild-type. When transfected in GH4C1 ce lls, the T149A receptor mutant fully inhibited basal cAMP and partiall y inhibited G(s)-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 calci um channel agonist (+/-)-Bay K8644, whereas the wild-type 5-HT1A recep tor inhibited the calcium influx by 40%. Thus, the Thr149 residue is d irectly involved in G protein coupling to calcium mobilization (mediat ed by beta gamma subunits of G(i2)) and to inhibition of calcium chann el activation (mediated by beta gamma subunits of G(o)) but plays a mi nor role in coupling to alpha(1)-mediated inhibition of cAMP accumulat ion, The conserved i2 loop threonine may serve as a G protein contact site to direct the signaling specificity of multiple receptors.