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

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. 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. INTRODUCTIONCellular responses to agonists of G-protein-coupled receptors are rapidly attenuated in the continuous presence of agonist and desensitize to repeated application of agonist (1Hausdorff W.P. Caron M.G. Lefkowitz R.J. FASEB J. 1990; 4: 2881-2889Crossref PubMed Scopus (1080) Google Scholar, 2Lohse M.J. Biochim. Biophys. Acta. 1993; 1179: 171-188Crossref PubMed Scopus (400) Google Scholar). With time between challenges, responses and are the of to to desensitization the of cells, to and are the of the receptors and in the but of the receptors is of The mechanisms may of G-protein-coupled receptors. and by the kinase kinase receptor kidney epithelial and the agonists and desensitize in a The of these receptor of responses J. PubMed Google Scholar, PubMed Scopus Google Scholar, Google Scholar). the are to expose a tethered that and activates the receptor in irreversible PubMed Scopus Google Scholar). a of that is rapidly attenuated the irreversible mechanism of J. 1993; PubMed Google Scholar). of responses to the of new receptor J. PubMed Google of receptors is a mechanism of desensitization (1Hausdorff W.P. Caron M.G. Lefkowitz R.J. FASEB J. 1990; 4: 2881-2889Crossref PubMed Scopus (1080) Google Scholar, 2Lohse M.J. Biochim. Biophys. Acta. 1993; 1179: 171-188Crossref PubMed Scopus (400) Google Scholar). and the to the and the The and the desensitize by this mechanism Google Scholar, J. Caron M.G. Lefkowitz R.J. J. 1993; PubMed Google Scholar, Google Scholar, J. Lefkowitz R.J. J. PubMed Google Scholar). endocytosis of receptors to desensitization by receptors the to agonists in the extracellular J. PubMed Google Scholar, PubMed Scopus Google Scholar, J. J. PubMed Scopus Google the and internalization J. PubMed Google Scholar, PubMed Scopus Google Scholar). receptor internalization and of agonist and and receptor and receptor Google Scholar, Lefkowitz R.J. W.P. J. 1993; PubMed Google Scholar, M.J. Google Scholar). the which is irreversibly activated by cleavage, is and in J. PubMed Google Scholar, J. 1993; PubMed Google Scholar, J. PubMed Google Scholar). of responses to mobilization of the large stores of the in the Golgi is the of the of receptors J. PubMed Scopus Google Scholar, J. J. PubMed Scopus Google Scholar, J. J. PubMed Scopus Google Scholar, J. PubMed Scopus Google Scholar). Trypsin cleaves PAR-2 extracellular a tethered that the receptor and activates J. PubMed Scopus Google Scholar, J. PubMed Scopus Google Scholar). PAR-2 is expressed in the and may activated by pancreatic and in cells, may to J. PubMed Scopus Google Scholar, J. PubMed Scopus Google Scholar). the PAR-2 is expressed by epithelial and may of the of trypsin J. PubMed Scopus Google Scholar). the of pancreatic by a mechanism FASEB J. PubMed Scopus Google and of and PubMed Scopus Google Scholar, of the Scholar, of the Scholar). of PAR-2 pancreatic of and J. PubMed Scopus Google Scholar, J. PubMed Scopus Google Scholar). The mechanisms of desensitization and of these are but are to of in the are episodically exposed to trypsin and of the Scholar). the of the may of in of PAR-2, of of this was to the mechanisms of desensitization and of PAR-2 in kidney epithelial and in cells, a the intestinal that naturally PAR-2. We (a) PAR-2-mediated Ca2+ (b) examined cleavage of PAR-2 by desensitization to repeated of PAR-2, the of in Ca2+ examined internalization and targeting of activated PAR-2, and the mechanism of of Ca2+ mechanisms of desensitization and of PAR-2-mediated responses are but are of the mechanism of irreversible of this receptor. It is to these desensitization of PAR-2 the of by and is to to to Thus, the of trypsin that are by PAR-2, which may of pancreatic and J. PubMed Scopus Google Scholar, J. PubMed Scopus Google are PAR-2 desensitization and resensitization. the of the to trypsin may of of J. J. PubMed Scopus Google Scholar). examined the mechanism of desensitization and of expressing PAR-2 to trypsin and these in epithelial human PAR-2 and that naturally express PAR-2. the mechanisms of desensitization and of the expressed in these PubMed Scopus Google Scholar, Google Scholar, J. PubMed Scopus Google to PAR-2 a receptor. of may to the of receptor Thus, cells, a the intestinal PubMed Scopus Google Scholar, J. Google Scholar). naturally PAR-2 J. PubMed Scopus Google and epithelial of the a is to desensitization and of PAR-2 in intestinal epithelial cells, are episodically exposed to trypsin and of the Ca2+ and Ca2+ examined the of PAR-2 by trypsin Ca2+i, Trypsin and stimulated increase in in and trypsin was trypsin trypsin and in and of trypsin of the Ca2+ response was a rapid increase in Ca2+i, and a to a sustained which of this response was due to mobilization of Ca2+ intracellular stores and which of extracellular Ca2+, and in cells, trypsin stimulated a prompt increase in Ca2+i, which rapidly to Ca2+ was to the to a of exposure to cells, the response to trypsin rapidly in to cells, the was not but Ca2+ was to a to and in of trypsin and in and in a in Trypsin was and in and in to which Ca2+ was to a of in the extracellular and and in to which Ca2+ was to a of in the extracellular are to the in the of and that was the influx of extracellular Ca2+. of to and the influx of Ca2+ which the the influx in and that the prompt increase in that of PAR-2 is due to of Ca2+ intracellular was in the of extracellular Ca2+. of the response is rapidly attenuated the irreversible of PAR-2 The of the and the mechanism of are the the the influx of extracellular Ca2+, in was in the of extracellular Ca2+. that in cells, in to influx of extracellular Ca2+, Ca2+ mobilization intracellular stores to the The influx of extracellular Ca2+ is by of the intracellular Ca2+ which activates the the that is inhibited by PubMed Scopus Google Scholar). Ca2+ the extracellular in response to PAR-2 the in Thus, PAR-2 is to the and which to agonists by intracellular Ca2+ and by a in the that is to Google Scholar). The influx of Ca2+ extracellular is of Ca2+ influx the intracellular the of of Ca2+ and is of the of by may a in J. J. PubMed Scopus Google Scholar, PubMed Scopus Google Scholar). may PAR-2, and which naturally express PAR-2, Ca2+ responses to trypsin and and PAR-2 of J. PubMed Scopus Google and of is the of a response to repeated exposure to agonist to presence of responses to trypsin that are by PAR-2 desensitize by (a) cleavage of the which that a receptor by (b) receptor and and of of intracellular signaling. activates PAR-2 receptor cleavage, that responses to desensitize by and of of signaling. desensitization of PAR-2 trypsin and Ca2+ responses to repeated application of trypsin and examined desensitization of PAR-2 by to trypsin cells, trypsin the Ca2+ response to a exposure to trypsin and but not the Ca2+ response to a exposure to but not the response to cells, trypsin the response to a exposure to trypsin and and the response to a exposure to and markedly inhibited the response to Thus, of PAR-2 by trypsin desensitization of the Ca2+ Trypsin a in of the Ca2+ response in trypsin exposed to are of the Ca2+ response of and to and exposed to exposed to not exposed to exposed to exposed to trypsin trypsin exposed to exposed to exposed to trypsin trypsin exposed to exposed to exposed to trypsin trypsin exposed to not exposed to trypsin trypsin exposed to exposed to in a new of the Ca2+ response in trypsin exposed to are Ca2+ in and We of PAR-2 trypsin caused desensitization of responses to by to and then a of trypsin and the response to and and this was the Thus, of PAR-2 may desensitization of responses in the Ca2+ response to in was of that in cells, that receptors. may the in desensitization of the response in these by trypsin and The intracellular Ca2+ stores not by trypsin was to Ca2+. that desensitization of the Ca2+ response to trypsin and is not due to of these of stores to a examined Ca2+ responses to and cells, the response to but not the response to trypsin the response desensitization and desensitization of that repeated application of trypsin marked desensitization of PAR-2. due to of the receptor of intracellular was of Ca2+. Trypsin a desensitization of PAR-2 the of the Ca2+ response to agonists is is but not cleaves PAR-2, which that a receptor to We the of this cleavage by of the and of PAR-2 the the of expressing PAR-2 We exposed to and the followed by a to trypsin resulted in a rapid of to of that in and of exposure to resulted in a of to of min. of trypsin due to receptor cleavage and receptor receptor internalization We examined the of receptor cleavage and receptor endocytosis to of of exposure to was in and in in of exposure to was in and in in and the of to We that the of trypsin is by receptor cleavage and is by receptor We that these internalization of the examined in a in the PubMed Scopus Google endocytosis may to desensitization by receptors the to a that is to agonists in the extracellular We examined this by desensitization of the Ca2+ response to of trypsin in and to cells, trypsin to the Ca2+ response to a exposure to trypsin the response to a desensitization to trypsin is by of receptor and internalization is not the mechanism of rapid desensitization of PAR-2. that was desensitization of PAR-2-mediated Ca2+ mobilization the between application of trypsin was this time was internalization of PAR-2 in which that endocytosis is not the mechanism of desensitization. We that internalization of the is not by this receptor is rapidly agonist PubMed Scopus Google Scholar, Google of desensitization of the Ca2+ response of and to trypsin and not not in a new the Ca2+ to Trypsin and desensitization of G-protein-coupled receptors is by receptor and (1Hausdorff W.P. Caron M.G. Lefkowitz R.J. FASEB J. 1990; 4: 2881-2889Crossref PubMed Scopus (1080) Google Scholar, 2Lohse M.J. Biochim. Biophys. Acta. 1993; 1179: 171-188Crossref PubMed Scopus (400) Google Scholar). in this and the receptor to the between the receptor and and desensitization. in desensitization to desensitization by receptors that desensitization by receptors in of is to of which and the of Ca2+ intracellular which the influx of extracellular activates J. PubMed Scopus Google Scholar, J. PubMed Scopus Google Scholar, M.J. 1993; PubMed Scopus Google Scholar). desensitization of PAR-2 by receptor and by of mechanisms. We and to the of to desensitization of PAR-2-mediated Ca2+ the of of the trypsin the of the Ca2+ response to trypsin and in a and The was and the response was was to of the Thus, of the of the Ca2+ response to trypsin and in and of the Ca2+ response to trypsin in are of of the Ca2+ response to trypsin and in and of of the Ca2+ response to trypsin and in and and and and and and exposure to trypsin in and are expressed a of the response and are of the of of by trypsin and cells, inhibited the and of the Ca2+ response to trypsin and in a and The was and the response was by exposed to of the and to of the the of the this was caused by of We that of the Ca2+ response to trypsin and in and is to the which is the influx of extracellular Ca2+ in in response to We examined this in exposed to trypsin and then the of the Ca2+ was a in the response was by of but by of We that the which is activated by PAR-2, is by of the Ca2+ response to trypsin in are of the Ca2+ response to in the of the Ca2+ response are the that is the of Ca2+ by intracellular in increase in of intracellular Ca2+ stores by the influx of extracellular Ca2+ the that is inhibited by PubMed Scopus Google Scholar). the of and PAR-2-mediated Ca2+ mobilization due to a the of these the Ca2+ response to stimulated a prompt and sustained increase in in The of the response was in but Ca2+ was to extracellular which that the is due to influx of extracellular Ca2+. was by that Ca2+ the to the response to the increase in by was by the of in the influx of extracellular Ca2+, exposed to and the of the Ca2+ the the was of the response to the was of the to and in the was of the Thus, of is activated by (PAR-2) to desensitization of receptors to the response to agonist the a in the intracellular that is to (1Hausdorff W.P. Caron M.G. Lefkowitz R.J. FASEB J. 1990; 4: 2881-2889Crossref PubMed Scopus (1080) Google Scholar, 2Lohse M.J. Biochim. Biophys. Acta. 1993; 1179: 171-188Crossref PubMed Scopus (400) Google Scholar). PAR-2 a in the intracellular PAR-2 is not to to examined to desensitization of PAR-2. of to the Ca2+ response to trypsin of by of not the trypsin Thus, that desensitization of that in PAR-2-mediated Ca2+ is and desensitization of G-protein-coupled receptors. Thus, Ca2+ mobilization and by receptors J. 1993; PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, J. 1993; PubMed Google Scholar, 1993; Google Scholar, PubMed Scopus Google the PubMed Scopus Google Scholar). that of the Ca2+ response to trypsin is by the that responses to and PubMed Scopus Google Scholar, PubMed Scopus Google Scholar). It is that of the of and of of the mechanism in of receptors are not the of the the and the receptor are not agonist J. Lefkowitz R.J. J. PubMed Google Scholar, J. J. 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The that of the a rapid that the desensitization in the intracellular of human and human PAR-2 that the and in the that are in PAR-2 in the intracellular that is a a the of in the of PAR-2 and the marked of and that may a in desensitization of PAR-2 of the is that and to the (1Hausdorff W.P. Caron M.G. Lefkowitz R.J. FASEB J. 1990; 4: 2881-2889Crossref PubMed Scopus (1080) Google Scholar, 2Lohse M.J. Biochim. Biophys. Acta. 1993; 1179: 171-188Crossref PubMed Scopus (400) Google may in the to PAR-2-mediated Ca2+ that the is not the of is the that which J. PubMed Scopus Google is not in response to the is J. 1993; PubMed Google Scholar). and 1993; Google that the of the Ca2+ response to which to is not to and is not by is a Ca2+ mobilization and of and is in response to of by a that J. 1990; PubMed Google Scholar). the may mechanism of desensitization J. 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Acta. 1993; 1179: 171-188Crossref PubMed Scopus (400) Google Scholar). to that PAR-2 by trypsin but that this is not the mechanism of rapid desensitization. endocytosis may to a of desensitization by the of receptors that are to agonists in the extracellular this of PAR-2 in by and trypsin and and PAR-2 was the to the exposure to PAR-2 was the in a and in that the exposed to trypsin PAR-2 was in that between the and the and was a of the of the PAR-2 was in that the a caused internalization of PAR-2 into and into the Thus, activated PAR-2 is rapidly the of PAR-2 in exposure to trypsin and and PAR-2 was by the and a is a the presence of PAR-2 in the in a and in the and the of and the of PAR-2 in not exposed to the and to to the Golgi PAR-2 was in a of the and that by the was by of a of The that PAR-2 not of disrupted the Golgi of PAR-2 and and in that the PAR-2 and not in of The this is but that the of PAR-2 and by that the of in the and Golgi PubMed Scopus Google Scholar). Thus, of the and Golgi to and and of the in kidney epithelial PubMed Scopus Google Scholar). the of and of the Golgi is by by to the intracellular of PAR-2 and the of the of PAR-2 and and and in PAR-2 and by the a and a and a The in was and is a of The in was exposed to and is a is a of and is a of and the presence of PAR-2 the and in the Golgi that the Golgi PAR-2 and the PAR-2 that trypsin early PAR-2 and the exposure to trypsin this of the PAR-2 of the and that the receptor was by of time was of PAR-2 and the receptor Thus, PAR-2 but is then into the of PAR-2 the receptor in trypsin and PAR-2 was the receptor by the a and a receptor and a is a of and is a The the marked of PAR-2 and the receptor. is the is the irreversible cleavage and by that PAR-2 not but to activates PAR-2 receptor cleavage, that the receptor PAR-2 is irreversible and PAR-2 and a and trypsin to and to of PAR-2 and of receptor PAR-2 the exposure to trypsin and of these and of that was the PAR-2 was in the of the and Thus, PAR-2 is to and this the receptor is irreversibly activated by trypsin activated by the of PAR-2 and the and in trypsin and PAR-2 was by the a and a a is a of and is a of and is a of The that of the large that PAR-2 are lysosomes. that PAR-2 by trypsin rapidly and then to is degraded. We not PAR-2 is in cells, to the receptor. the is in and in that naturally express this receptor J. PubMed Google Scholar, J. 1993; PubMed Google Scholar). the is by and in and is then to J. PubMed Google Scholar, J. 1993; PubMed Google Scholar, J. PubMed Google Scholar, J. Google Scholar). are to to and to J. 1993; PubMed Google Scholar). Thus, the targeting of the receptors is that of the receptors and The and rapidly agonists into early endosomes and in endosomes J. PubMed Google Scholar, PubMed Scopus Google Scholar). receptors are to lysosomes. The this in between the receptors and is but receptor are to the targeting of receptors in the and of G-protein-coupled receptors and receptors are endocytosis 1993; PubMed Scopus Google Scholar). PAR-2 The of in intracellular and may a receptors and J. 1993; PubMed Scopus Google targeting to may We not the of these in PAR-2, this of PAR-2 and the PAR-2 is to cleavage trypsin this targeting is of the mechanism of and the of PAR-2, which is by cleavage, is not in in intracellular between receptors and receptors that resensitize by to Trypsin and by of PAR-2 and by of is to to to may the of trypsin which may of PAR-2 J. PubMed Scopus Google Scholar). the time of desensitization and of PAR-2-mediated Ca2+ mobilization to repeated trypsin exposed to a of and then a of trypsin the and cells, to trypsin the response to a of trypsin the to of that in and The response resensitized to the between trypsin was min. in the Ca2+ response to a of the between was and was min. the of the desensitization to repeated exposure to trypsin was desensitization to repeated exposure to of repeated application of trypsin the Ca2+ response in and exposed to a of trypsin and exposed to a of trypsin of are expressed a of the response and are the mechanism of of PAR-2 to repeated application of trypsin by of protein synthesis and receptor and by Golgi of PAR-2. We exposed to a of trypsin and then a of trypsin exposed to of trypsin of and then a of trypsin the The Ca2+ response to a of trypsin resensitized to of that of by exposure to of trypsin the of the stores of PAR-2 in the Golgi was A, which to the stores of PAR-2 in Brefeldin to of trypsin but markedly inhibited to of trypsin to of new protein synthesis was cycloheximide, which protein synthesis in PubMed Scopus Google Scholar). to of trypsin but inhibited to of trypsin to of We that which and of the in cells, of this receptor PubMed Scopus Google Scholar, Google Scholar). to was PAR-2 to of trypsin of repeated application of trypsin the Ca2+ response in and exposed to a of trypsin and exposed to a of trypsin 60-90 exposed to of trypsin 60-90 and exposed to a of trypsin 60-90 the trypsin and trypsin to of in a exposure to The of A, cycloheximide, and are expressed a of the response and are a examined of PAR-2 in of The Ca2+ response to a of trypsin resensitized by a of trypsin but resensitized to of by exposure to of Brefeldin of trypsin but markedly inhibited of in of and that to trypsin of PAR-2, in and a of the Golgi stores A, that the stores of PAR-2 are not disrupted by stores may a of PAR-2 in a PAR-2 was in in to trypsin was markedly attenuated by that the stores are by the of and Golgi are the stores of PAR-2 that are to to trypsin was attenuated by cycloheximide, that the synthesis of PAR-2 is to receptors of receptor cleavage and in exposure to trypsin in but not cells, that new receptor synthesis is in between the is that was of to which may of intracellular of PAR-2 of new PAR-2 synthesis to the receptors. which is and of receptors PubMed Scopus Google Scholar, Google is not of PAR-2, not resensitization. of responses to is of intracellular stores of the and new protein synthesis J. PubMed Google Scholar). the PAR-2 resensitizes by intracellular of receptor and by synthesis of new receptors. new protein synthesis is not of the PubMed Scopus Google Scholar). are intracellular of the in in PubMed Scopus Google Scholar, J. PubMed Scopus Google Scholar). receptor resensitizes by is inhibited by Google is expressed in in the and J. PubMed Scopus Google Scholar, J. PubMed Scopus Google Scholar). the PAR-2 is expressed in epithelial cells, in the of the PAR-2 is the of these cells, may activated by pancreatic trypsin in the intestinal and of the of trypsin in the the of pancreatic by trypsin FASEB J. PubMed Scopus Google and of trypsin and of the Scholar, of the Scholar). trypsin is in and between Thus, PAR-2 activated the by in and in cells, which intestinal epithelial in that PAR-2 rapidly desensitize by a of (a) cleavage, which irreversible of PAR-2, and (b) which may receptor and of the which the trypsin is epithelial to PAR-2 by intracellular and by synthesis of new receptors. of the mechanisms of desensitization and of PAR-2 in of the and of INTRODUCTIONCellular responses to agonists of G-protein-coupled receptors are rapidly attenuated in the continuous presence of agonist and desensitize to repeated application of agonist (1Hausdorff W.P. Caron M.G. Lefkowitz R.J. FASEB J. 1990; 4: 2881-2889Crossref PubMed Scopus (1080) Google Scholar, 2Lohse M.J. Biochim. Biophys. Acta. 1993; 1179: 171-188Crossref PubMed Scopus (400) Google Scholar). With time between challenges, responses and are the of to to desensitization the of cells, to and are the of the receptors and in the but of the receptors is of The mechanisms may of G-protein-coupled receptors. and by the kinase kinase receptor kidney epithelial and the agonists and desensitize in a The of these receptor of responses J. PubMed Google Scholar, PubMed Scopus Google Scholar, Google Scholar). the are to expose a tethered that and activates the receptor in irreversible PubMed Scopus Google Scholar). a of that is rapidly attenuated the irreversible mechanism of J. 1993; PubMed Google Scholar). of responses to the of new receptor J. PubMed Google of receptors is a mechanism of desensitization (1Hausdorff W.P. Caron M.G. Lefkowitz R.J. FASEB J. 1990; 4: 2881-2889Crossref PubMed Scopus (1080) Google Scholar, 2Lohse M.J. Biochim. Biophys. Acta. 1993; 1179: 171-188Crossref PubMed Scopus (400) Google Scholar). and the to the and the The and the desensitize by this mechanism Google Scholar, J. Caron M.G. Lefkowitz R.J. J. 1993; PubMed Google Scholar, Google Scholar, J. Lefkowitz R.J. J. PubMed Google Scholar). endocytosis of receptors to desensitization by receptors the to agonists in the extracellular J. PubMed Google Scholar, PubMed Scopus Google Scholar, J. J. PubMed Scopus Google the and internalization J. PubMed Google Scholar, PubMed Scopus Google Scholar). receptor internalization and of agonist and and receptor and receptor Google Scholar, Lefkowitz R.J. W.P. J. 1993; PubMed Google Scholar, M.J. Google Scholar). the which is irreversibly activated by cleavage, is and in J. PubMed Google Scholar, J. 1993; PubMed Google Scholar, J. PubMed Google Scholar). of responses to mobilization of the large stores of the in the Golgi is the of the of receptors J. PubMed Scopus Google Scholar, J. J. PubMed Scopus Google Scholar, J. J. PubMed Scopus Google Scholar, J. PubMed Scopus Google Scholar). Trypsin cleaves PAR-2 extracellular a tethered that the receptor and activates J. PubMed Scopus Google Scholar, J. PubMed Scopus Google Scholar). PAR-2 is expressed in the and may activated by pancreatic and in cells, may to J. PubMed Scopus Google Scholar, J. PubMed Scopus Google Scholar). the PAR-2 is expressed by epithelial and may of the of trypsin J. PubMed Scopus Google Scholar). the of pancreatic by a mechanism FASEB J. PubMed Scopus Google and of and PubMed Scopus Google Scholar, of the Scholar, of the Scholar). of PAR-2 pancreatic of and J. PubMed Scopus Google Scholar, J. PubMed Scopus Google Scholar). The mechanisms of desensitization and of these are but are to of in the are episodically exposed to trypsin and of the Scholar). the of the may of in of PAR-2, of of this was to the mechanisms of desensitization and of PAR-2 in kidney epithelial and in cells, a the intestinal that naturally PAR-2. We (a) PAR-2-mediated Ca2+ (b) examined cleavage of PAR-2 by desensitization to repeated of PAR-2, the of in Ca2+ examined internalization and targeting of activated PAR-2, and the mechanism of of Ca2+