Agonist‐induced long‐term desensitization of the human prostacyclin receptor

Abstract

Phosphorylation of the human prostacyclin (PGI 2 ) receptor (hIP-R) by diacylglycerol-regulated protein kinase C (PKC) has been reported to be responsible for its rapid desensitization in HEK293 cells. In this study we demonstrate, that human fibroblasts reveal a much slower hIP-R desensitization kinetics, which was neither affected by stimulation nor inhibition of PKC by either phorbol 12-myristate-13-acetate or GF-109203X suggesting a different cellular mechanism. Although agonist-promoted sequestration of a C-terminally green fluorescent protein-tagged hIP-R was demonstrated, it did not account for the long-term desensitization. Concanavalin A did not abolish, but accelerated receptor desensitization kinetics. Resensitization of hIP-R involved receptor recycling and/or de novo synthesis of receptor protein, depending on the duration of prior desensitization. This is the first study investigating the mechanisms of hIP-R desensitization in intact human cells naturally expressing hIP-R. Our data suggest, that a hitherto unknown mechanism of hIP-R long-term desensitization, which is independent of receptor phosphorylation by conventional and novel type PKC isoforms or endocytosis, is a key event in regulating the cellular responsiveness to PGI 2 . ß

Recycling of the human prostacyclin receptor is regulated through a direct interaction with Rab11a GTPase

“…Hence, understanding the mechanisms whereby the cellular responses to prostacyclin are dynamically regulated is critical to the understanding of its role in such processes at the detailed molecular level. While much is known about the modes of intracellular signalling by the prostacyclin receptor (IP) and the events leading to its desensitization and internalization, significantly less is understood about the intracellular sorting mechanisms dictating its recyclization or other trafficking following agonist stimulation [14,[32][33][34][35]46]. Herein, we have uncovered a novel direct association between the hIP and the GTPase Rab11a that regulates its recycling to the plasma membrane following agonist stimulation.…”

Regulation of Thromboxane Receptor Trafficking Through the Prostacyclin Receptor in Vascular Smooth Muscle Cells

“…Rapid recycling, and hence restoration of responsiveness, is readily observed for GPCRs that traffic in an arrestinindependent manner. 10,13,30 In the case of the IP, its postendocytotic fate appears dependent on cell type: rapid recycling has been reported in platelets, 29 fibroblasts, 28 and transfected HEK293 cells, 10 whereas a slower process, dependent on de novo protein synthesis, contributes to restoration of membrane IP expression in some cells, including vascular SMCs. 29,31 Neither homologous internalization of TP ( Figure 3C), nor its postendocytotic processing ( Figure 5C), were modified overtly when IP and TP␣ were coexpressed.…”

Internalization and Recycling of the Human Prostacyclin Receptor Is Modulated through Its Isoprenylation-dependent Interaction with the δ Subunit of cGMP Phosphodiesterase 6

“…Once internalized the receptor may be dephosphorylated and recycled to the cell surface, restoring rapidly agonist activation, or undergo lysosomal degradation resulting in receptor down-regulation and a sustained loss of responsiveness (4). While the regulatory mechanisms employed by many GPCRs adhere, to a reasonable degree, to this pathway, we (5,6) and others (7,8) have reported that, similar to other GPCRs (9,10), regulation of IP deviates from this norm. Thus rapid desensitization of activated IP occurs secondary to it's phosphorylation, not by a GRK but by a second messenger activated kinase, PKC (6).…”

“…Subsequently the IP is internalized through a phosphorylation-and arrestin-independent pathway (5,7,8). Rapid recycling of the sequestered IP upon agonist withdrawal, which restores the response to agonist without the need for de novo protein synthesis, is evident in platelets (8), fibroblasts (7), and transfected HEK293 cells (5).…”