The 5-HT 7 receptors are encoded by a single gene and the gene transcript undergoes an alternative splicing leading to the generation of at least four different
S- palmitoylation is a reversible attachment of palmitate or other saturated long chain fatty acids to the target proteins. The attachment of the fatty acids occurs at the
3. MATERIALS AND METHODS
4.5. Role of the Palmitoylation in the Coupling of the Receptor with G-proteins
To test for the functional significance of the 5-HT1A receptor palmitoylation, we analysed the interaction of the 5-HT1A receptor with the Gα subunits of the heterotrimeric G-proteins by using the GTPγS coupling assay (Ponimaskin et al., 1998). First, the Gα-subunits were co-expressed with the wild type receptor in Sf.9 cells (in all cases the appropriate Gα-subunit was co-expressed with the β1 and the γ2 subunits), and the agonist-promoted binding of [35S] GTPγS to the Gα-subunit was assessed by counting radioactivity directly after immunoprecipitation with the antibodies directed against the appropriate Gα subunit (Fig. 4.6 A). When the wild type 5-HT1A receptor was co-expressed with Gαi1, Gαi2 or Gαi3, we measured a 1.7- to 2.5-fold increase in the [35S]
GTPγS binding upon stimulation with 1 µM 5-HT. The result confirmed that the 5-HT1A
receptor effectively communicates with the G-proteins of the Gi family. In contrast, there was no coupling after co-expression of the receptor with the Gαs, the Gα12 or the Gα13
subunits (Fig. 4.6 A).
We then tested an ability of the palmitoylation-deficient receptor mutants to couple with the Gαi3 protein. In the case of the single mutants C417-S and C420-S, the agonist-dependent GTPγS binding was significantly decreased, when compared to the WT receptor. However, some significant activation of the Gαi protein over the basal level was still detectable (Figure 4.6 B). In contrast, when the non-palmitoylated receptor mutant C417/420-S was expressed, the relative activation of the Gαi3 subunit after the agonist stimulation was completely abolished (Fig. 4.6 B). It is notable that the mutants as well as the WT 5-HT1A receptor were expressed in a similar level, as assessed by the Western blot analysis (Fig. 4.6 B, inset). To exclude the possibility that this effect was mediated by the change in the ligand binding properties, we performed the pharmacological analysis of the pharmacological of the wild type and the mutant 5-HT1A receptors expressed in the Sf.9 cells. We found that the pharmacological profile for the WT receptor was similar to that previously reported for the 5-HT1A receptor expressed in the Sf.9 cells (Clawges et al., 1997). More importantly, the analysis of the palmitoylation-deficient mutants revealed that the replacement of the palmitoylated cysteines did not change their pharmacological properties (Fig. 4.7; Tab. 4.1). The binding affinity of [3H]
5-HT for the wild type 5-HT1A receptor was similar to that obtained for the mutants.
Taken together, these data indicate a functional importance of the 5-HT1A receptor palmitoylation for the coupling to the Gi3-protein.
Figure 4.6 Effect of Palmitoylation on the Coupling between the 5-HT1A Receptor and Gi -Protein. (A) Communication of the 5-HT1A receptor with different G-proteins. Membranes were prepared from the Sf.9 cells expressing the recombinant proteins as indicated and then incubated with [35S] GTPγS in the presence of either H2O (vehicle) or 5-HT (1 µM). The immunoprecipitations were performed with the appropriate antibodies directed against the indicated Gα-subunits. (B) The membranes were isolated from the Sf.9 cells co-expressing the recombinant Gαi3- and the Gβ1γ2 -subunits together with either the 5-HT1A receptor wild type or the acylation-deficient mutants. After the incubation with [35S] GTPγS in the presence of H2O (vehicle) or 1 µM 5-HT, the membranes were lysed and the Gαi3-subunit was immunoprecipitated with the specific antibodies. The values obtained for the 5-HT1A receptor wild type after the agonist stimulation were set to 100%. The data points represent the means ± S.E. (n=6). A statistically significant difference between values is noted (*, p < 0.01). (Inset) Expression analysis for the WT and the acylation-deficient mutants. Samples from the parallel infections were used for the Western blot analysis with the Gαi3- or the HA-specific antibodies.
Figure 4.6 Effect of Palmitoylation on the Coupling between the 5-HT
A
GTPγS binding (fold increase overbasal level) 0.5
GTPγS binding (fold increase overbasal level) 0.5
GTPγS binding (fold increase overbasal level) 0.5 -Protein. (A) Communication of the 5-HT1A receptor with different G-proteins. Membranes were prepared from the Sf.9 cells expressing the recombinant proteins as indicated and then incubated with [35S] GTPγS in the presence of either H2O (vehicle) or 5-HT (1 µM). The immunoprecipitations were performed with the appropriate antibodies directed against the indicated Gα-subunits. (B) The membranes were isolated from the Sf.9 cells co-expressing the recombinant Gαi3- and the Gβ1γ2 -subunits together with either the 5-HT1A receptor wild type or the acylation-deficient mutants. After the incubation with [35S] GTPγS in the presence of H2O (vehicle) or 1 µM 5-HT, the membranes were lysed and the Gαi3-subunit was immunoprecipitated with the specific antibodies. The values obtained for the 5-HT1A receptor wild type after the agonist stimulation were set to 100%. The data points represent the means ± S.E. (n=6). A statistically significant difference between values is noted (*, p < 0.01). (Inset) Expression analysis for the WT and the acylation-deficient mutants. Samples from the parallel infections were used for the Western blot analysis with the Gαi3- or the HA-specific antibodies.
A
GTPγS binding (fold increase overbasal level) 0.5
GTPγS binding (fold increase overbasal level) 0.5
GTPγS binding (fold increase overbasal level) 0.5
Figure 4.7. Mutation of the Palmitoylation Sites Does Not Change the Ligand-Binding Properties of the 5-HT1A Receptor. Saturation binding of [3H] 5-HT with the WT and the palmitoylation-deficient 5-HT1A receptors was performed on the membranes prepared from infected Sf.9 cells. The non-specific binding did not exceed 5% of the specific binding and was subtracted from the total counts. Finally, the data were fitted to the one-site saturation model. Data points represent the means ± S.E. from at least three independent experiments performed in triplicate (n=9).
0,0
Table 4.1. Pharmacological Properties of the Palmitoylation-Deficient 5-HT1A Receptor Mutants Determined by the Saturation Binding of [3H] 5-HT to the Membranes of the Sf.9 Cells. The Kd and the Bmax values were calculated by the one-site saturation fit. Data are expressed in the means ± S. E.
(n=9)
Table 4.1. Pharmacological Properties of the Palmitoylation-Deficient 5-HT1A Receptor Mutants Determined by the Saturation Binding of [3H] 5-HT to the Membranes of the Sf.9 Cells. The Kd and the Bmax values were calculated by the one-site saturation fit. Data are expressed in the means ± S. E.
(n=9)
(pmol/mg protein)