CELL-SPECIFIC PHYSICAL AND FUNCTIONAL COUPLING OF HUMAN 5-HT(1A) RECEPTORS TO INHIBITORY G-PROTEIN ALPHA-SUBUNITS AND LACK OF COUPLING TO G(S-ALPHA)

We have studied the physical and functional linkages of heterologously expressed human 5-HT1A receptors to G protein alpha-subunits in HeLa and CHO-K1 cells. HeLa cells expressed immunoreactivity to G(i) proteins with an apparent rank order of G(ialpha3) (almost-equal-to 1 pmol/mg of protein) >> G(ialpha1) (almost-equal-to 0.1 pmol/mg) >> G(ialpha2) (< 0.02 pmol/mg), whereas CHO-K1 cells expressed immunoreactivity to G(ialpha2) (almost-equal-to 5 pmol/mg) >> G(ialpha3) (almost-equal-to 0.7 pmol/mg), but not to G(ialpha1). Both cell lines expressed large and small forms of G(salpha), but neither expressed detectable G(oalpha). Agonist-promotable physical coupling of the 5-HT1A receptor to G proteins was examined with high-affinity agonist binding and with co-immunoprecipitation using rabbit anti-receptor IgG fractions. Agonist treatment induced coupling of the 5-HT1A receptors to G proteins with an apparent rank order of G(ialpha3) > G(ialpha1), G(ialpha2) in HeLa cells and G(ialpha3) > G(ialpha2) in CHO-K1 cells. Agonist-promotable functional coupling of the 5-HT1A receptors to inhibition of adenylylcyclase was measured in membranes derived from HeLa and CHO-K1 cells expressing approximately 2.5-3 pmol of receptors/mg of protein by preincubation with antisera raised against the carboxyl termini of the G(i) protein alpha-subunits. A noteworthy difference between the two cell types was that antisera against the predominant G protein (G(ialpha2)) were substantially more efficacious than G(ialpha3) antisera at blocking functional coupling to adenylylcyclase inhibition in CHO-K1 cells, whereas in HeLa cells, antisera against nonpredominant G proteins (G(ialpha1)/G(ialpha2)) were equally as effective as those against the predominant G protein (G(ialpha3)). No physical or functional coupling of the 5-HT1A receptor to G(salpha) isoforms was detected in either cell line. These findings suggest that the 5-HT1A receptor can physically couple to multiple distinct G(i) proteins in mammalian cell membranes and that functional coupling to adenylylcyclase inhibition may be mediated by G(ialpha1), G(ialpha2), and G(ialpha3). One factor influencing the relative importance of those G proteins for 5-HT1A receptor-inhibited adenylylcyclase activity appears to be their relative levels of expression. Therefore, these results suggest that the coupling of the 5-HT1A receptor to adenylylcyclase relies in part on factors other than its rank order of affinity for G(i) protein alpha-subunits. It is likely that receptor/G protein molar ratios are important in determining the specificity of G protein coupling of the 5-HT1A receptor.