Mechanisms of Desensitization and Resensitization of Proteinase-activated Receptor-2

doi:10.1074/jbc.271.36.22003

Mechanisms of Desensitization and Resensitization of Proteinase-activated Receptor-2*

From the Departments of ^‡ Surgery,
^¶ Medicine, and
^∥ Physiology, University of California, San Francisco, California 94143-0660 and the
^§ Department of Nutritional Sciences, University of Calfornia, Berkeley, California 94720

** To whom correspondence should be addressed:
Box 0660, University of California, San Francisco, 521 Parnassus Ave., San Francisco, CA 94143-0660.
Tel.: 415-476-0489; Fax: 415-476-0936.

Abstract

Proteinase-activated receptor-2 (PAR-2) is a G-protein-coupled receptor that is expressed by intestinal epithelial cells, which are episodically exposed to pancreatic trypsin in the intestinal lumen. Trypsin cleaves PAR-2 to expose a tethered ligand, which irreversibly activates the receptor. Thus, PAR-2 may desensitize and resensitize by novel mechanisms. We examined these mechanisms in kidney epithelial cells, stably expressing human PAR-2, and intestinal epithelial cells, which naturally express PAR-2. Trypsin stimulated a prompt increase in [Ca²⁺]_i, due to mobilization of intracellular Ca²⁺, followed by a sustained plateau, due to influx of extracellular Ca²⁺. Repeated application of trypsin caused marked desensitization of this response, which is due in part to (a) irreversible cleavage of the receptor by trypsin and (b) protein kinase C-mediated termination of signaling. Trypsin exposure resulted in internalization of PAR-2 into early endosomes and then lysosomes; but endocytosis was not the mechanism of rapid desensitization. Thus, activated PAR-2 is endocytosed and degraded. The Ca²⁺ response to trypsin resensitized by 60-90 min. Brefeldin A, which disrupted Golgi stores of PAR-2, and cycloheximide, which inhibited protein synthesis, markedly attenuated resensitization. Thus, PAR-2-mediated Ca²⁺ mobilization desensitizes by irreversible receptor cleavage, protein kinase C-mediated termination of signaling, and PAR-2 targeting to lysosomes. It resensitizes by mobilization of large Golgi stores and synthesis of new receptors.

Footnotes

↵* This work was supported by National Institutes of Health Grants DK43207, DK46285, DK39957, and NS21710. The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

β₂-AR

β₂-adrenergic receptor

PAR-2

proteinase-activated receptor-2

Th-R

thrombin receptor

NK1-R

neurokinin 1 receptor

AP

activating peptide

PKC

protein kinase C

PKA

protein kinase A

GRK

G-protein receptor kinase

KNRK

sarcoma virus transformed rat kidney epithelial cells

PDBu

phorbol 12,13-dibutyrate.
- Received April 3, 1996.
- Revision received June 5, 1996.