Specificity of heterotrimeric G protein regulation by human chorionic gonadotropin and low-molecular agonist of luteinizing hormone receptor

Luteinizing hormone (LH) and its homologue, human chorionic gonadotropin (hCG), are very important regulators of the reproductive system. These hormones stimulate various types of G proteins—primarily, G_s and G_q proteins—by binding to the specific LH-hCG receptor, which leads to the activation of adenylate cyclase (AC) and phospholipase C, respectively. It has been suggested that many side effects of LH and hCG are associated with low selectivity of their effect on G proteins. Low-molecular agonists of LH-hCG receptor developed on the basis of thienopyrimidine derivatives do not cause these side effects, and differences in the interaction with G proteins may be ones of the cause for this. To test this, a comparative study of the effect of hCG and synthesized by us thienopyrimidine derivative, 5-amino-N-tert-butyl-2-(methylsulfanyl)-4-(3-(nicotinamido)phenyl)thieno[2,3-d]pyrimidine-6-carboxamide (TP03) on the AC activity and GTP binding of G proteins in plasma membranes isolated from the rat ovaries and testes was performed. Cholera toxin (CT) and pertussis toxin (PT) were used to selectively switch off the signal transduction via G_s and G_i/o proteins, the peptide corresponding to the C-terminal segment 349–359 of the Gα_q subunit was used to suppress G_q-dependent cascades. It was shown that treatment of ovarian and testicular membranes with CT resulted in suppression of TP03 and hCG stimulatory effects on the AC activity, but in different ways influenced the GTP binding stimulation: it completely blocked the effect of 10^–6 M TP03 and reduced by 45–46% the effect of hCG (10^–8 M). Preincubation of membranes with the peptide 349–359 reduced the hCG stimulatory effect on GTP binding by 34 (ovaries) and 45% (testes), but did not affect the corresponding effect of 10^–6 M TP03. Preincubation with the peptide 349–359 also reduced the GTP stimulatory effect of 10^–4 M TP03, but to a small extent. The obtained data indicate that, in contrast to hCG, the targets of which in the ovaries and testes are G_s and G_q proteins, the action of TP03 is realized mainly via G_s proteins. Only at a concentration that exceeds EC₅₀ by two orders TP03 is capable to relatively weakly activate G_q proteins. The PT treatment of the membranes did not affect the effects of TP03 and hCG, which indicates the lack of their effective interaction with G_i/o proteins. Thus, the selectivity of activation of G_s-dependent cascades responsible for the synthesis and production of steroid hormones is a significant advantage of low-molecular agonists of LH-hCG receptor over gonadotropins.