Characterization of the G-protein coupling domains of the 5-HT1A receptor
Creators: name: Kushwaha, Neena
Publisher: name: Université d'Ottawa / University of Ottawa
Year: 2007
Subjects: subject: Biology, Molecular.

description: The 5-HT1A receptor is expressed pre-synaptically as the primary somatodendritic autoreceptor on serotonergic raphe neurons, and post-synaptically in several brain regions. However, many of the 5-HT1A-mediated signaling pathways initially identified in non-neuronal cells transfected with the receptor have not been extensively studied in neuronal models. Our first study focused on signaling of the 5-HT1A autoreceptor in RN46A cells, a model of serotonergic raphe neurons that express endogenous 5-HT1A receptors. We found that in RN46A cells stably transfected with the wild-type 5-HT1A receptor, 5-HT1A receptor activation coupled to Galphai-mediated inhibition of forskolin-induced cAMP formation, Gbetagamma-mediated increase in [Ca2+]I, and induced a novel inhibition of phospho-p42/p44-mitogen activated protein kinase (MAPK) that was also Gbetagamma-dependent. The activity of 5-HT1A receptors containing mutations of PKC sites in the second (i2: T149A) or third intracellular loop (i3: T229A/S253G/T343A) was also tested. The signaling of the 5-HT1A i3 mutant was similar to the 5-HT1A wild-type receptor while the i2 and quadruple (i2/i3) mutants, which both comprise the T149A substitution, failed to couple to Gbetagamma-mediated increase in [Ca2+]I or inhibition of MAPK, but did couple to Galphai-mediated inhibition of cAMP. Thus, the i2 domain of the 5-HT1A autoreceptor is crucial for coupling to Gbetagamma-mediated signaling. Based on these findings and previous studies performed in our lab, we wanted to investigate the structure-function relationship of the T149 amino acid and adjacent residues in the coupling of G-proteins, in particular Gbetagamma subunits, to the 5-HT1A receptor. Using a random mutagenesis approach, over 60 mutant 5-HT1A receptors were generated in the alpha-helical Ci2 loop sequence (143DYVNKRTPRR152) and tested for functionality. Mutant receptors were tested for Gbetagamma signaling to adenylyl cyclase II (ACII) or phospholipase C (PLC) and GalphaI coupling using sensitive assays to detect constitutive and agonist-induced coupling. Our data support an amphipathic alpha-helical model coiled at Pro150, with the positively charged face oriented cytoplasmically. The uncharged residues (T149, N146) in this face are required for Gbetagamma but not GalphaI coupling and may directly interact with Gbetagamma subunits. The hydrophobic face is oriented internally towards the C-terminal i3 (Ci3) and N-terminal i2 (Ni2) loops, and includes the critical Y144 that directs the specificity of coupling to both Gbetagamma and GalphaI pathways. Coordinate hydrogen and ionic bonding between functionally key residues Y144/K147 (Ci2), D133/R134 (Ni2 DRY motif), and E340 (Ci3) is predicted to stabilize the G-protein coupling domain. These results provide the first detailed evidence that the entire Ci2 domain is a critical determinant for receptor coupling to G-proteins, especially Gbetagamma subunits, to induce multiple responses.