Characterisation of palmitoylation in alpha₂_A adrenoceptor and 5-HT₁_A serotonin receptor-G₀₁α G protein fusion proteins

Abstract

Palmitoylation variant GPCR-G protein fusion proteins were created between the porcine u2A-adrenoceptor or the human 5-HT1A-serotonin receptor and the pertussis toxin resistant, Cys35lIle, form of the rat Go1u protein. These palmitoylation-variant fusions were transiently expressed in HEK293T cells prior to analysis of the regulation of palmitoylation and the functional consequences of palmitoylation for both the GPCR and G protein parts of the fusions. When the regulation of palmitoylation was studied for u2A-adrenoceptor-GoluCys35IIle fusion proteins, dynamic palmitoylation and depalmitoylation of both the Cys442residue of the u2A-adrenoceptor and the Cys ' residue of the GoluCys351Ile protein were found to occur. However, only the GOluCys351Ileprotein part of the fusion was found to undergo adrenaline-stimulated regulation of palmitoylation and the effect of adrenaline required G protein activation. Adrenaline regulation proceeded in a concentration-dependent manner correlating with agonist occupancy of the u2A-adrenoceptor. Such agonist effects were found to be, at least in part, due to agonist-stimulation of GOluCys351Ile protein depalmitoylation. The requirements for palmitoylation of the u2A-adrenoceptor and GoluCys351Ile protein elements of the u2A-adrenoceptor-GoluCys35IUe fusion proteins were subsequently assessed for various functional properties. Palmitoylation of neither the U2Aadrenoceptor nor the GoluCys351Ile protein parts of the fusion determined fusion protein expression levels, affinity for the agonist adrenaline, affinity for the antagonist RS- 79948-197, ability to bind or to hydrolyse GTP or their ability to influence the efficiency of RGS 16 protein to accelerate the GTPase reaction. In regulation of palmitoylation studies for 5-HTIA-receptor-GoluCys35IIle fusion proteins, dynamic palmitoylation of the Cys' residue of the GoluCys351Ue protein and the Cys417 residue of the 5-HTIA-receptor was observed as well as a lack of incorporation of palmitate into Cys420 of the 5-HT1A-receptor. Dynamic depalmitoylation was only observed for the Cys' residue of the GoluCys351Ile protein, not for the 5-HT1A-receptor. In the latter case, palmitate once incorporated appeared to remain stably attached. Both the 5-HT1A-receptor and the GoluCys351Ile protein parts of the fusion were found to undergo 8-0H-DPAT-stimulated regulation ofpalmitoylation. 8-0H-DPAT was able to regulate palmitoylation levels of both proteins in a concentration-dependent manner. For the regulation of GoluCys351Ile protein palmitoylation such agonist effects were found likely to be, at least in part, due to an agonist-stimulated rate of depalmitoylation. For the regulation of 5-HT1A-receptor palmitoylation such agonist-stimulated increases in observed palmitoylation levels were only attributable to the addition of palmitate, given that no depalmitoylation of the 5- HT1A-receptor could be detected. The requirements for palmitoylation of the 5-HT1A-receptor and GoluCys351Ile protein elements of the 5-HT1A-receptor-GoluCys351Ile fusion proteins were also assessed for a selection of functional properties. Similar to the results obtained with Go1uCys351Ile protein constrained to the uZA-adrenoceptor, the palmitoylation of the GoluCys351Ile protein did not determine fusion protein expression levels, their affinity for the antagonist WAYI00635, or their ability to bind GTP. Palmitoylation of 5-HT1Areceptor did not alter fusion protein expression levels or their affinity for the antagonist WAYI00635. However, in contrast, it did cause enhanced levels of GTP binding to the 5-HT1A-receptor-GoluCys351Ile fusion proteins. The results of this investigation suggest that there are different requirements for regulation of GPCR and G protein palmitoylation dependent on the GPCR-G protein fusion in question. These requirements may be responsible for the specific functional properties displayed by such fusions. The current study also demonstrates that GPCR-G protein fusion proteins can be successfully used as tools to study both the regulation of palmitoylation and the functional consequences of this modification