Differential coupling of 5-HT(1) receptors to G proteins of the G(i) family

Abstract

1: Since all 5-HT(1) receptors couple to G(i)-type G proteins and inhibit adenylyl cyclase, the functional significance of five distinct subtypes of 5-HT(1) receptors has been unclear. 2: In previous studies we have used transfected cells to demonstrate that 5-HT(1B) receptors can couple more efficiently than 5-HT(1A) receptors to activation of extracellular signal-regulated kinase (ERK) and to inhibition of adenylyl cyclase. These findings suggested the possibility that individual 5-HT(1) receptors differentially couple to isoforms of G(ialpha). 3: In the present study we utilized a model system in which pertussis toxin resistant forms of human G(ialpha1), G(ialpha2), and G(ialpha3) were used to directly compare the coupling of human 5-HT(1A), 5-HT(1B), and 5-HT(1D) receptors to each G(ialpha) in transfected human HeLa cells. 4: 5-HT(1A) receptors displayed a preference for G(ialpha1) and G(ialpha2), relative to G(ialpha3). Pertussis toxin resistant forms of G(ialpha1), G(ialpha2), and G(ialpha3) rescued 73%, 76%, and 44%, respectively, of the ERK activation stimulated by 5-HT in the absence of pertussis toxin. 5: In contrast, pertussis toxin resistant forms of G(ialpha1), G(ialpha2), and G(ialpha3) rescued 32%, 118%, and 35% of 5-HT(1B) receptor-stimulated activity, respectively, indicating that 5-HT(1B) receptors coupled primarily through G(ialpha2). A similar preference for G(ialpha2) was found in studies of the 5-HT(1D) receptor, where toxin resistant G(ialpha1), G(ialpha2), and G(ialpha3) rescued 30%, 70%, and 40% of activity, respectively. 6: In conclusion, the observed differential coupling of 5-HT(1) receptors to isoforms of G(ialpha), provides additional evidence for our previous findings that the subtypes of 5-HT(1) receptors exhibit similar, but distinct, functions.

Figure 1

5-HT_1A receptors couple to activation of ERK through pertussis toxin sensitive G proteins. (A) Nontransfected HeLa cells were treated for 5 min with 10 μM 5-HT (lane 2), and then lysed. (B) HeLa cells transfected with cDNA for the human 5-HT_1A receptor were treated overnight in the presence (lanes 3 and 4) or absence (lanes 1 and 2) of 20 ng ml⁻¹ pertussis toxin before treatment with 10 μM 5-HT (lanes 2 and 4) for 5 min, and subsequent lyses. Total lysate was analysed by immunoblotting with antibody to phospho-ERK1/ERK2 (p-ERK). Membranes were then stripped and analysed with antibody to total ERK1/ERK2 (Total). Net intensities of bands were calculated from three separate experiments, performed in duplicate, and expressed as the means±s.e.mean (×10³). *P<0.05; ***P<0.001; n.s., statistically not significant vs absence of 5-HT, two-sided paired Student t-test calculated separately for both the presence and absence of pertussis toxin. Representative immunoblots from one of the three experiments are shown to demonstrate that treatment with pertussis toxin does not alter the levels of total ERK.

Figure 2

5-HT_1A receptors couple to activation of ERK through multiple subtypes of G_iα. HeLa cells co-transfected with cDNA for the human 5-HT_1A receptor and pertussis toxin resistant forms of either (A) G_iα1, (B) G_iα2, or (C) G_iα3 were treated overnight in the presence (lanes 3 and 4) or absence (lanes 1 and 2) of 20 ng ml⁻¹ pertussis toxin before treatment with 10 μM 5-HT (lanes 2 and 4) for 5 min, and subsequent lyses. Total lysate was analysed by immunoblotting with antibody to phospho-ERK1/ERK2 (p-ERK). Membranes were then stripped and analysed with antibody to total ERK1/ERK2 (Total). Net intensities of bands were calculated from three separate experiments, performed in duplicate, and expressed as the means±s.e.mean (×10³). **P<0.01; ***P<0.001 vs absence of 5-HT, two-sided paired Student t-test calculated separately for both the presence and absence of pertussis toxin. Representative immunoblots from one of the three experiments are shown to demonstrate that treatment with pertussis toxin does not alter the levels of total ERK.

Figure 3

5-HT_1A receptors couple most efficiently to activation of ERK through G_iα2 despite equal expression of all transfected G_iα subtypes. (A) HeLa cells co-transfected with cDNA for the human 5-HT_1A receptor and pertussis toxin resistant forms of either G_iα1, G_iα2, or G_iα3 were treated overnight with 20 ng/ml pertussis toxin before treatment with the indicated concentrations of 5-HT for 5 min, and subsequent lyses. (B) HeLa cells were transfected with cDNA for pertussis toxin resistant forms of either G_iα1 (lane 2), G_iα2 (lane 3), or G_iα3 (lane 4) and the density of expression of G_iα subunits were compared to nontransfected (con) cells (lane 1). (A) Total lysate was analysed by immunoblotting with antibody to phospho-ERK1/ERK2 (p-ERK) or (B) 20 μg of total lysate was analysed by immunoblotting with antibody recognizing all forms of G_iα. Net intensities of bands were calculated from three separate experiments, performed in duplicate, and expressed as the means±s.e.mean (×10³) **P<0.01; ***P<0.001 vs nontransfected (con) cells, ANOVA, Bonferroni analysis. A representative immunoblot from one of the three experiments is shown.

Figure 4

5-HT_1B and 5-HT_1D receptors couple to activation of ERK through pertussis toxin sensitive G proteins. (A) HeLa cells transfected with cDNA for the human 5-HT_1B receptor or (B) human 5-HT_1D receptor were treated overnight in the presence (lanes 3 and 4) or absence (lanes 1 and 2) of 20 ng ml⁻¹ pertussis toxin before treatment with 10 μM 5-HT (lanes 2 and 4) for 5 min, and subsequent lyses. Total lysate was analysed by immunoblotting with antibody to phospho-ERK1/ERK2 (p-ERK). Membranes were then stripped and analysed with antibody to total ERK1/ERK2 (Total). Net intensities of bands were calculated from three separate experiments, performed in duplicate, and expressed as the means±s.e.mean (×10³). *P<0.05; **P<0.01; n.s., statistically not significant vs absence of 5-HT, two-sided paired Student t-test calculated separately for both the presence and absence of pertussis toxin. Representative immunoblots from one of the three experiments are shown to demonstrate that treatment with pertussis toxin does not alter the levels of total ERK.

Figure 5

5-HT_1B receptors couple preferentially to G_iα2. HeLa cells co-transfected with cDNA for the human 5-HT_1B receptor and pertussis toxin resistant forms of either (A) G_iα1, (B) G_iα2, or (C) G_iα3 were treated overnight in the presence (lanes 3 and 4) or absence (lanes 1 and 2) of 20 ng ml⁻¹ pertussis toxin before treatment with 10 μM 5-HT (lanes 2 and 4) for 5 min, and subsequent lyses. Total lysate was analysed by immunoblotting with antibody to phospho-ERK1/ERK2 (p-ERK). Membranes were then stripped and analysed with antibody to total ERK1/ERK2 (Total). Net intensities of bands were calculated from three separate experiments, performed in duplicate, and expressed as the means±s.e.mean (×10³). *P<0.05; **P<0.01; ***P<0.001 vs absence of 5-HT, two-sided paired Student t-test calculated separately for both the presence and absence of pertussis toxin. Representative immunoblots from one of the three experiments are shown to demonstrate that treatment with pertussis toxin does not alter the levels of total ERK.

Figure 6

5-HT_1D receptors are similar to 5-HT_1B receptors in preferentially coupling to G_iα2. HeLa cells co-transfected with cDNA for the human 5-HT_1D receptor and pertussis toxin resistant forms of either (A) G_iα1, (B) G_iα2, or (C) G_iα3 were treated overnight in the presence (lanes 3 and 4) or absence (lanes 1 and 2) of 20 ng ml⁻¹ pertussis toxin before treatment with 10 μM 5-HT (lanes 2 and 4) for 5 min, and subsequent lyses. Total lysate was analysed by immunoblotting with antibody to phospho-ERK1/ERK2 (p-ERK). Membranes were then stripped and analysed with antibody to total ERK1/ERK2 (Total). Net intensities of bands were calculated from three separate experiments, performed in duplicate, and expressed as the means±s.e.mean (×10³). *P<0.05; **P<0.01; ***P<0.001 vs absence of 5-HT, two-sided paired Student t-test calculated separately for both the presence and absence of pertussis toxin. Representative immunoblots from one of the three experiments are shown to demonstrate that treatment with pertussis toxin does not alter the levels of total ERK.