Homologous desensitization of human histamine H3 receptors expressed in CHO-K1 cells

- Histamine H3 receptors (H3Rs) modulate several nervous system functions.
- Homologous desensitization was tested for human H3Rs (hH3Rs) expressed in CHO-K1 cells.
- hH3R binding and functional responses were markedly reduced by agonist pre-incubation.
- hH3Rs desensitize and internalize in a GRK2- and clathrin-dependent manner.
- Desensitization is a relevant issue for pharmacological therapies aimed at H3Rs.

Histamine H3 receptors (H3Rs) modulate the function of the nervous system at the pre- and post-synaptic levels. In this work we aimed to determine whether, as other G protein-coupled receptors (GPCRs), H3Rs desensitize in response to agonist exposure. By using CHO-K1 cells stably transfected with the human H3R (hH3R) we show that functional responses (inhibition of forskolin-induced cAMP accumulation in intact cells and stimulation of [35S]-GTPγS binding to cell membranes) were markedly reduced after agonist exposure. For cAMP accumulation assays the effect was significant at 60 min with a maximum at 90 min. Agonist exposure resulted in decreased binding sites for the radioligand [3H]-N-methyl-histamine ([3H]-NMHA) to intact cells and modified the sub-cellular distribution of H3Rs, as detected by sucrose density gradients and [3H]-NMHA binding to cell membranes, suggesting receptor internalization. The reduction in the inhibition of forskolin-stimulated cAMP formation observed after agonist pre-incubation was prevented by incubation in hypertonic medium or in ice-cold medium. Agonist-induced loss in binding sites was also prevented by hypertonic medium or incubation at 4 °C, but not by filipin III, indicating clathrin-dependent endocytosis. Immunodetection showed that CHO-K1 cells express GPCR kinases (GRKs) 2/3, and both the GRK general inhibitor ZnCl2 and a small interfering RNA against GRK-2 reduced receptor desensitization. Taken together these results indicate that hH3Rs experience homologous desensitization upon prolonged exposure to agonists, and that this process involves the action of GRK-2 and internalization via clathrin-coated vesicles.