Cell-Type Specific Effects of Endocytosis Inhibitors on 5-Hydroxytryptamine_2AReceptor Desensitization and Resensitization Reveal an Arrestin-, GRK2-, and GRK5-Independent Mode of Regulation in Human Embryonic Kidney 293 Cells

Abstract

The effect of endocytosis inhibitors on 5-hydroxytryptamine(2A) (5-HT(2A)) receptor desensitization and resensitization was examined in transiently transfected human embryonic kidney (HEK) 293 cells and in C6 glioma cells that endogenously express 5-HT(2A) receptors. In HEK-293 cells, 5-HT(2A) receptor desensitization was unaffected by cotransfection with a dominant-negative mutant of dynamin (DynK44A), a truncation mutant of arrestin-2 [Arr2(319-418)], or by two well-characterized chemical inhibitors of endocytosis: concanavalin A (conA) and phenylarsine oxide (PAO). In contrast, beta 2-adrenergic receptor desensitization was significantly potentiated by each of these treatments in HEK-293 cells. In C6 glioma cells, however, DynK44A, Arr2(319-418), conA, and PAO each resulted in the potentiation of 5-HT(2A) and beta-adrenergic receptor desensitization. The cell-type-specific effect of Arr2(319-418) on 5-HT(2A) receptor desensitization was not related to the level of GRK2 or GRK5 expression. Interestingly, although beta 2-adrenergic receptor resensitization was potently blocked by cotransfection with DynK44A, 5-HT(2A) receptor resensitization was enhanced, suggesting the existence of a novel cell-surface mechanism for 5-HT(2A) receptor resensitization in HEK-293 cells. In addition, Arr2(319-418) had no effect on 5-HT(2A) receptor resensitization in HEK-293 cells, although it attenuated the resensitization of the beta 2-adrenergic receptor. However, in C6 glioma cells, both DynK44A and Arr2(319-418) significantly reduced 5-HT(2A) receptor resensitization. Taken together, these results provide the first convincing evidence of cell-type-specific roles for endocytosis inhibitors in regulating GPCR activity. Additionally, these results imply that novel GRK and arrestin-independent mechanisms of 5-HT(2A) receptor desensitization and resensitization exist in HEK-293 cells.

Identification of Two Serine Residues Essential for Agonist-Induced 5-HT2AReceptor Desensitization

“…Thus, in initial experiments, we compared phorbol ester-and agonist-induced 5-HT 2A receptor desensitization. As shown in Figure 2A, pretreatment with quipazine, a 5-HT 2A receptor agonist, induces a maximum of 65% desensitization with a t 1/2 for desensitization of 42 min, in agreement with prior studies (8,10). In comparison, pretreatment with phorbol 12,13-dibutyrate (PDBu), a PKC activator, results in an immediate reduction in the level of 5-HT 2A receptor signaling, terminating to a level similar to that for agonistpretreated cells (Figure 2A).…”

“…fetal calf serum. Agonist-mediated desensitization was performed exactly as previously described (10). To assess phorbol 12,13-dibutyrate (PDBu)-mediated 5-HT 2A receptor desensitization, cells were preincubated for the indicated times in the absence or presence of 1 µM PDBu then washed with inositol-free DMEM.…”

“…5-HT 2A receptors are known to desensitize following agonist exposure (8)(9)(10); thus, the stimulatory effect of 5-HT will be brief and not repeated during high-frequency stimulation. These apparent limits on 5-HT 2A receptor signaling, the high concentration of 5-HT required for receptor activation and the rapid attenuation of signaling, suggest that prolonged or repeated stimulation of 5-HT 2A receptors, possibly due to a failure in the desensitization process, could be pathological.…”

Aripiprazole, A Novel Atypical Antipsychotic Drug with a Unique and Robust Pharmacology

“…PI hydrolysis experiments were performed as previously described, with minor changes Gray et al, 2001;Shapiro et al, 2000). Briefly, PO1C, GF62, or C6-glioma cells were grown in T75 flasks to 80% confluency, and plated into 24-well plates with Dulbecco's modified Eagle's medium containing 10% dialyzed fetal calf serum.…”

Pharmacologic analysis of non-synonymous coding h5-HT2A SNPs reveals alterations in atypical antipsychotic and agonist efficacies

“…Indeed, the activity/function of 5-HT 2A receptors is known to be affected by the cellular context (e.g., cellular G-protein complement, receptor interacting proteins) in which the receptor is expressed. 40,41 Two studies 42,43 examined intracellular, 5-HT-induced calcium release in platelets. In one study of 16 seasonal affective disorder patients, Ozaki et al 42 reported a significantly smaller rise in 5-HT-stimulated intracellular calcium flux in 452 heterozygote (His452/Tyr452) cells compared to His452 homozygote cells.…”