The Adenosine 2b Receptor Is Recruited to the Plasma Membrane and Associates with E3KARP and Ezrin upon Agonist Stimulation

Abstract

We have previously shown that adenosine is formed in the intestinal lumen during active inflammation from neutrophil-derived 5′-AMP. Acting through the adenosine A2b receptor (A2bR), the luminally derived adenosine induces vectorial chloride secretion and a polarized secretion of interleukin-6 to the intestinal lumen. Although some G protein-coupled receptors interact with anchoring or signaling molecules, not much is known in this critical area for the A2bR. We used the model intestinal epithelial cell line, T84, and Caco2-BBE cells stably transfected with GFP-A2b receptor to study the intestinal A2bR. The A2bR is present in both the apical and basolateral membranes of intestinal epithelia. Apical or basolateral stimulation of the A2bR induces recruitment of the receptor to the plasma membrane and caveolar fractions. The A2bR co-immunoprecipitates with E3KARP and ezrin upon agonist stimulation. Ezrin interacts with E3KARP and PKA and the interaction between ezrin and E3KARP is enhanced by agonist stimulation. Our data suggest that the A2bR is recruited to the plasma membrane upon apical or basolateral agonist stimulation and interacts with E3KARP and ezrin. We speculate that such an interaction may not only anchor the A2bR to the plasma membrane but may also function to stabilize the receptor in a signaling complex in the plasma membrane. We have previously shown that adenosine is formed in the intestinal lumen during active inflammation from neutrophil-derived 5′-AMP. Acting through the adenosine A2b receptor (A2bR), the luminally derived adenosine induces vectorial chloride secretion and a polarized secretion of interleukin-6 to the intestinal lumen. Although some G protein-coupled receptors interact with anchoring or signaling molecules, not much is known in this critical area for the A2bR. We used the model intestinal epithelial cell line, T84, and Caco2-BBE cells stably transfected with GFP-A2b receptor to study the intestinal A2bR. The A2bR is present in both the apical and basolateral membranes of intestinal epithelia. Apical or basolateral stimulation of the A2bR induces recruitment of the receptor to the plasma membrane and caveolar fractions. The A2bR co-immunoprecipitates with E3KARP and ezrin upon agonist stimulation. Ezrin interacts with E3KARP and PKA and the interaction between ezrin and E3KARP is enhanced by agonist stimulation. Our data suggest that the A2bR is recruited to the plasma membrane upon apical or basolateral agonist stimulation and interacts with E3KARP and ezrin. We speculate that such an interaction may not only anchor the A2bR to the plasma membrane but may also function to stabilize the receptor in a signaling complex in the plasma membrane. protein kinase A cystic fibrosis transmembrane conductance regulator adenosine 2b receptor green fluorescent protein Chinese hamster ovary 5′-ectonucleotidase Hank's balanced salt solution Adenosine is an ubiquitous extracellular signaling molecule that is released during inflammation and acts as a paracrine factor with diverse effects on the inflammatory cascade (1Olah M.E. Stiles G.L. Annu. Rev. Pharmacol. Toxicol. 1995; 35: 581-606Crossref PubMed Google Scholar, 2Feoktistov I. Biaggioni I. J. Clin. Invest. 1995; 96: 1979-1986Crossref PubMed Scopus (309) Google Scholar, 3Feoktistov I. Biaggioni I. Pharmacol. Rev. 1997; 49: 381-402PubMed Google Scholar, 4Linden J. Auchampach J.A. Xiaowei J. Figler R.A. Life Sci. 1995; 62: 1519-1524Crossref Scopus (45) Google Scholar, 5Roman R.M. Fitz J.G. Gastroenterologia. 1999; 116: 964-979Abstract Full Text Full Text PDF Scopus (113) Google Scholar, 6Van Belle H. Gossens F. Wynants J. Am. J. Physiol. 1987; 252: H886-H893PubMed Google Scholar, 7Zidek Z. Eur. Cytokine Netw. 1999; 10: 319-328PubMed Google Scholar). In the intestine, neutrophil transmigration into the lumen to form crypt abscesses is the pathologic hallmark of the active phase of many intestinal disorders, including inflammatory bowel disease. We have previously shown that neutrophils, upon transmigration into the intestinal lumen, release 5′-AMP (8Nash S. Parkos C. Nusrat A. Delp C. Madara J.L. J. Clin. Invest. 1991; 87: 1474-1477Crossref PubMed Scopus (65) Google Scholar, 9Madara J.L. Patapoff T.W. Gillece-Castro B. Colgan S. Parkos C. Delp C. Mrsny R. J. Clin. Invest. 1993; 91: 5716-5723Crossref Scopus (238) Google Scholar). Adenosine is derived from 5′-AMP when it is converted by the intestinal apical membrane 5′-ectonucleotidase (CD 73) (11Strohmeier G.R. Lencer W.I. Thompson L.F. Carlson S.L. Moe S.J. Carnes D.K. Mrsny R.J. Madara J.L. J. Clin. Invest. 1997; 99: 2588-2601Crossref PubMed Scopus (145) Google Scholar) and can subsequently interact with the intestinal adenosine receptor (10Strohmeier G.R. Reppert S.M. Lencer W.I. Madara J.L. J. Biol. Chem. 1995; 270: 2387-2394Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar). The adenosine receptor belongs to the family of the G protein-coupled group of cell surface receptors (12Palmer T.M. Stiles G.L. Neuropharmacology. 1995; 34: 683-694Crossref PubMed Scopus (265) Google Scholar, 13Ralevic V. Burnstock G. Pharmacol. Rev. 1998; 50: 413-492PubMed Google Scholar). On the basis of initial pharmacological criteria, adenosine is known to interact with one of the four adenosine receptor subtypes A1, A2a, A2b, and A3 (3Feoktistov I. Biaggioni I. Pharmacol. Rev. 1997; 49: 381-402PubMed Google Scholar, 4Linden J. Auchampach J.A. Xiaowei J. Figler R.A. Life Sci. 1995; 62: 1519-1524Crossref Scopus (45) Google Scholar, 12Palmer T.M. Stiles G.L. Neuropharmacology. 1995; 34: 683-694Crossref PubMed Scopus (265) Google Scholar,13Ralevic V. Burnstock G. Pharmacol. Rev. 1998; 50: 413-492PubMed Google Scholar), all of which have now been cloned. Using molecular, pharmacologic, and biochemical approaches we characterized the intestinal adenosine receptor as the A2b subtype in both T84 cells, a model intestinal epithelial cell line, and intact human intestinal epithelia (10Strohmeier G.R. Reppert S.M. Lencer W.I. Madara J.L. J. Biol. Chem. 1995; 270: 2387-2394Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar). Furthermore, the A2bR appears to be the only adenosine receptor present in T84 cells (10Strohmeier G.R. Reppert S.M. Lencer W.I. Madara J.L. J. Biol. Chem. 1995; 270: 2387-2394Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar, 14Christofi F.L. Zhang H., Yu, J.G. Guzman J. Xue J. Kim M. Wang Y.Z. Cooke H.J. J. Comp. Neurol. 2001; 439: 46-64Crossref PubMed Scopus (111) Google Scholar). In these cells, the A2bR is functionally coupled to Gαs and the stimulation of the apical or basolateral surface with adenosine results in cAMP-mediated vectorial chloride secretion (9Madara J.L. Patapoff T.W. Gillece-Castro B. Colgan S. Parkos C. Delp C. Mrsny R. J. Clin. Invest. 1993; 91: 5716-5723Crossref Scopus (238) Google Scholar, 10Strohmeier G.R. Reppert S.M. Lencer W.I. Madara J.L. J. Biol. Chem. 1995; 270: 2387-2394Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar, 15Barrett K.E. Cohn J.A. Huott P.A. Wasserman S.I. Dharmsathaphorn K. Am. J. Physiol. 1990; 258: C902-C912Crossref PubMed Google Scholar, 16Barrett K.E. Huott P.A. Shah S. Dharmasathaphorn K. Am. J. Physiol. 1989; 256: C197-C203Crossref PubMed Google Scholar, 17Sitaraman S.V. Merlin D. Wang L. Wong M. Gewirtz A.T., Si- Tahar M. Madara J.L. J. Clin. Invest. 2001; 107: 861-869Crossref PubMed Scopus (159) Google Scholar) and interleukin-6 synthesis and secretion (18Sitaraman S.V., Si- Tahar M. Merlin D. Strohmeier G.R. Madara J.L. Am. J. Physiol. 2000; 278: C1230-C1236Crossref PubMed Google Scholar). The distribution and coupling of A2bR upon agonist stimulation with cytoplasmic structural proteins and the potential significance of this is not known. On the basis of effective coupling of minute second messenger signals to protein kinase A (PKA)1 we hypothesized sometime ago that A2bR might be compartmentalized with signaling molecules and their targets (10Strohmeier G.R. Reppert S.M. Lencer W.I. Madara J.L. J. Biol. Chem. 1995; 270: 2387-2394Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar). Huang et al. (19Huang P. Lazarowski E.R. Tarran R. Milgram S.L. Boucher R.C. Stutts M.J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 14120-14125Crossref PubMed Scopus (178) Google Scholar) recently provided functional evidence for such an organized complex including Gαs; adenylate cyclase, PKA, and CFTR are compartmentalized in microdomains in the apical plasma membrane. Such compartmentalization of signaling networks in a multiprotein complex has been shown to result in both the stabilization of constituent proteins at the cell surface and enhanced efficiency of signaling (20Sheng M. Sala C. Annu. Rev. Neurosci. 2001; 24: 1-29Crossref PubMed Scopus (1054) Google Scholar). A common mechanism for establishing multiple protein complexes is via protein-protein interaction with submembrane scaffolding proteins. PSD-95/Dlg/ZO-1 (PDZ) domain proteins localized to the membrane-cytoskeletal interface have emerged as important organizing centers for regulatory complexes, and these scaffold-based regulatory proteins are often polarized to specific sites in polarized epithelial cells (20Sheng M. Sala C. Annu. Rev. Neurosci. 2001; 24: 1-29Crossref PubMed Scopus (1054) Google Scholar, 21Sun F. Hug M.J. Lewarchik C.M. Yun C.H. Bradbury N.A. Frizzell R.A. J. Biol. Chem. 2000; 275: 29539-29546Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar). For example, ERM (ezrin-radixin-moesin)-binding protein (EBP50 or NHERF-1) and its related homolog NHE-3 kinase regulatory proteins (E3KARP or NHERF-2) appear to coordinate specific cAMP-mediated secretory responses (21Sun F. Hug M.J. Lewarchik C.M. Yun C.H. Bradbury N.A. Frizzell R.A. J. Biol. Chem. 2000; 275: 29539-29546Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar). The PDZ interacting motif (S/T)XL at the C terminus of proteins such as β-adrenergic receptor (22Hall R.A. Premont R.T. Chow C.W. Blitzer J.T. Pitcher J.A. Claing A. Stoffel R.H. Barak L.S. Shenolikar S. Weinman E.J. Grinstein S. Lefkowitz R.J. Nature. 1998; 392: 626-630Crossref PubMed Scopus (524) Google Scholar) and CFTR (23Sun F. Hug M.J. Bradbury N.A. Frizzell R.A. J. Biol. Chem. 2000; 275: 14360-14366Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar), binds to NHERF, which in turn interacts with ezrin. Ezrin is known to act as a protein kinase A anchoring protein (21Sun F. Hug M.J. Lewarchik C.M. Yun C.H. Bradbury N.A. Frizzell R.A. J. Biol. Chem. 2000; 275: 29539-29546Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar) in addition to associating with the actin cytoskeleton (reviewed in Ref. 24Bretscher A. Chambers D. R. D. Annu. Rev. Biol. 2000; PubMed Scopus Google Scholar). The interaction between NHERF, and PKA has been shown to be critical for the functional of including CFTR (21Sun F. Hug M.J. Lewarchik C.M. Yun C.H. Bradbury N.A. Frizzell R.A. J. Biol. Chem. 2000; 275: 29539-29546Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar) and NHE-3 C.H. G. A. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). In this study we the of A2bR at and stimulation with and We that the A2bR is recruited to the plasma membrane upon stimulation with The recruitment of the A2bR to the membrane is by its interaction with E3KARP and ezrin. Ezrin interacts with the A2bR and is in to adenosine stimulation. data suggest that the and its signaling in the in the and adenosine signaling through the A2bR may be by such interaction and recruitment of the receptor to the membrane. from Adenosine and from for and membranes from from from Yun and a from Thompson of from and from and from T84 cells and in as previously (8Nash S. Parkos C. Nusrat A. Delp C. Madara J.L. J. Clin. Invest. 1991; 87: 1474-1477Crossref PubMed Scopus (65) Google Scholar) in a of and with and by on cells for on of or with membrane in as previously (8Nash S. Parkos C. Nusrat A. Delp C. Madara J.L. J. Clin. Invest. 1991; 87: 1474-1477Crossref PubMed Scopus (65) Google Scholar). apical and basolateral membranes to be with apical and basolateral a to that previously for (8Nash S. Parkos C. Nusrat A. Delp C. Madara J.L. J. Clin. Invest. 1991; 87: 1474-1477Crossref PubMed Scopus (65) Google Scholar). The T84 cells a on T84 cells between and Caco2-BBE as in a of and with and cell in the surface with on cells in with and A2bR into the into the of the human A2bR and by to of the the The by the Caco2-BBE cells transfected with for in for the and in The from in and the by of fluorescent cell A2bR a from R. J. Mrsny of T84 or Caco2-BBE cells in in for with the in a with and subsequently with also with to by as (18Sitaraman S.V., Si- Tahar M. Merlin D. Strohmeier G.R. Madara J.L. Am. J. Physiol. 2000; 278: C1230-C1236Crossref PubMed Google Scholar). In the of Caco2-BBE cells transfected with with on and by Using actin the apical surface of the cell as and the basolateral surface at the of actin from the of the at from the the of and at the of actin used for of apical and basolateral with and and to the a to membranes and with membranes with and subsequently with to proteins with the to Apical or basolateral of the as previously Si- Tahar M. S.V. K. I. Madara J.L. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). The cell with to proteins. by and as previously Si- Tahar M. S.V. K. I. Madara J.L. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar) to plasma membrane and as E.J. C. Proc. Natl. Acad. Sci. U. S. A. 1995; PubMed Scopus Google Scholar) a T84 in with for at and with apical or basolateral adenosine A plasma membrane from The plasma membrane by this as previously not with or membranes from the of some proteins such as the of or G protein-coupled receptors is from the plasma membrane E.J. C. Proc. Natl. Acad. Sci. U. S. A. 1995; PubMed Scopus Google Scholar). with in or by and in transfected with A2bR with as D. S. K. J. B. Madara J.L. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). of on of cells from on the as by the Life with and and adenosine in the and an used for (10Strohmeier G.R. Reppert S.M. Lencer W.I. Madara J.L. J. Biol. Chem. 1995; 270: 2387-2394Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar). The of a of on the as J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). The of a and as the of at the for The for the The data are as the We characterized the to the second extracellular of the A2bR. T84 cells, which we have shown previously the an at In membrane from cells A2bR also a at The by used to the not cells, which of A2bR and to agonist at and as in cells transfected with A2bR an at to that in T84 cells and data that the the A2bR. We the of the A2bR. Using cell surface we that the A2bR is present at both the apical and basolateral membranes of the model intestinal epithelial as shown in the A2bR is at the basolateral membrane with the apical membrane an protein (11Strohmeier G.R. Lencer W.I. Thompson L.F. Carlson S.L. Moe S.J. Carnes D.K. Mrsny R.J. Madara J.L. J. Clin. Invest. 1997; 99: 2588-2601Crossref PubMed Scopus (145) Google Scholar), an of a protein Nusrat A. Parkos L. Carnes D. T.W. Madara J.L. 1997; PubMed Google Scholar) an of we the distribution of A2bR with data on cell surface the A2bR is both at the apical and basolateral surface of T84 cells The of the T84 epithelia at the apical and basolateral the cell surface of the of the at the apical and basolateral as in the of of A2b receptor with the and of and of and with by or also with the to the at and of the from apical and basolateral in the are at the of apical the of and basolateral of of the epithelial and through of the the A as and The the apical basolateral on its A2bR is to have a of data suggest that the A2bR in T84 cells is the of the A2bR in intestinal epithelial cells we stably transfected the intestinal cell line, with A2bR. shown in Caco2-BBE cells a in with cells a of We used an to the protein from cell and the A2bR on a in transfected cells a to the not present in the Caco2-BBE cells or in cells transfected with and The GFP-A2b protein enhanced the second and of cell of fluorescent and We the distribution of protein cell surface to the T84 cells, the A2bR present at both the apical and basolateral surface but at the basolateral surface to basolateral of from a an protein in Caco2-BBE cells (11Strohmeier G.R. Lencer W.I. Thompson L.F. Carlson S.L. Moe S.J. Carnes D.K. Mrsny R.J. Madara J.L. J. Clin. Invest. 1997; 99: 2588-2601Crossref PubMed Scopus (145) Google Scholar, J.A. J. PubMed Scopus Google Scholar), an of Nusrat A. Parkos L. Carnes D. T.W. Madara J.L. 1997; PubMed Google Scholar) an of from data not of transfected Caco2-BBE cells also the of the A2bR at the apical and basolateral surface the cell surface of the of the at the apical and basolateral The of of A2b receptor and an of and and data not the protein the cells with adenosine and stimulation. Apical or basolateral stimulation of cells with adenosine an in with cells transfected with data that Caco2-BBE cells transfected with A2bR has a as the A2bR in T84 the of the to the receptor in T84 cells, we the of agonist stimulation on the distribution of A2bR. Caco2-BBE cells are known to one of adenosine T84 cells in which A2bR is the only adenosine receptor present used to the A2bR. in a of A2bR in the plasma membrane in cells and the of the receptor present in the In receptor in the plasma membrane apical or basolateral stimulation with adenosine from The caveolar A2bR at and a but in this apical or basolateral stimulation from and in the caveolar in from plasma membrane and caveolar with the Apical or basolateral adenosine not have on the distribution of in these apical basolateral from data that apical or basolateral stimulation with adenosine results in the recruitment of A2bR to the plasma membrane to a to the caveolar and E3KARP are membrane PDZ domain proteins that may to anchor membrane proteins such as CFTR to the and E3KARP are to in with ezrin and PKA to a signaling complex in to membrane such as CFTR efficiency and proteins in the membrane. the PDZ domain protein may A2bR membrane we with A2bR. shown in E3KARP with the A2bR. apical or basolateral adenosine stimulation and in cells and The A2bR also with ezrin apical or basolateral stimulation for with adenosine The of the A2bR with ezrin receptor stimulation that of the in cells with apical or basolateral In as shown in apical or basolateral stimulation of the A2bR enhanced the interaction between E3KARP and ezrin with apical or basolateral the interaction of ezrin with PKA not by adenosine stimulation adenosine data that the A2bR interacts with E3KARP and ezrin. with the data in the ezrin with E3KARP and PKA The interaction between A2bR and ezrin and ezrin and E3KARP enhanced upon stimulation with In this study we used the human intestinal epithelial cell T84 to the distribution and of A2bR to its signaling in to agonist stimulation. The cell T84, previously used to of neutrophil-derived 5′-AMP to adenosine (CD A2bR as the only adenosine and its signaling mechanism subsequently shown to in human intestinal epithelia (9Madara J.L. Patapoff T.W. Gillece-Castro B. Colgan S. Parkos C. Delp C. Mrsny R. J. Clin. Invest. 1993; 91: 5716-5723Crossref Scopus (238) Google Scholar, 10Strohmeier G.R. Reppert S.M. Lencer W.I. Madara J.L. J. Biol. Chem. 1995; 270: 2387-2394Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar, G.R. Lencer W.I. Thompson L.F. Carlson S.L. Moe S.J. Carnes D.K. Mrsny R.J. Madara J.L. J. Clin. Invest. 1997; 99: 2588-2601Crossref PubMed Scopus (145) Google Scholar, 14Christofi F.L. Zhang H., Yu, J.G. Guzman J. Xue J. Kim M. Wang Y.Z. Cooke H.J. J. Comp. Neurol. 2001; 439: 46-64Crossref PubMed Scopus (111) Google Scholar). In this study we the of the The an at in T84 cells, cells transfected with and membranes A2bR. not present in cells or Caco2-BBE In GFP-A2b protein we that at to the Furthermore, and we that cells transfected with or A2bR also an at to that in T84 cells not results that the the A2bR. Our data is in with to in the that the A2bR a of C. V. F. P. J. C. R. Pharmacol. 2001; PubMed Scopus Google Scholar, F. I. F. C. 2000; PubMed Scopus Google Scholar) in human and cells transfected with the The A2bR is known to have and in a on 1995; PubMed Scopus Google Scholar). in protein to that in receptors such as receptor has been for the A2bR (3Feoktistov I. Biaggioni I. Pharmacol. Rev. 1997; 49: 381-402PubMed Google Scholar). may the in the in study with a of J. Figler H. Pharmacol. 1999; Google Scholar). We used the to the A2bR in the model intestinal epithelia. We and have previously shown that A2b is the only adenosine receptor present in T84 cells and in intact human intestinal epithelia (10Strohmeier G.R. Reppert S.M. Lencer W.I. Madara J.L. J. Biol. Chem. 1995; 270: 2387-2394Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar, 14Christofi F.L. Zhang H., Yu, J.G. Guzman J. Xue J. Kim M. Wang Y.Z. Cooke H.J. J. Comp. Neurol. 2001; 439: 46-64Crossref PubMed Scopus (111) Google Scholar, R. Madara J.L. Thompson L.F. Pharmacol. 1995; Google Scholar). we that the A2bR is present at both the apical and basolateral membranes of T84 surface a of the A2bR at the basolateral surface with apical We and have shown that for the of by basolateral stimulation with apical stimulation (10Strohmeier G.R. Reppert S.M. Lencer W.I. Madara J.L. J. Biol. Chem. 1995; 270: 2387-2394Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar, 15Barrett K.E. Cohn J.A. Huott P.A. Wasserman S.I. Dharmsathaphorn K. Am. J. Physiol. 1990; 258: C902-C912Crossref PubMed Google Scholar, 16Barrett K.E. Huott P.A. Shah S. Dharmasathaphorn K. Am. J. Physiol. 1989; 256: C197-C203Crossref PubMed Google Scholar). The receptor at the basolateral membrane may the to basolateral adenosine stimulation. Our data that apical or basolateral stimulation of the A2bR results in receptor in the plasma membrane Such recruitment and of receptor in and of the membrane is known to for G protein-coupled receptors including adenosine receptor P. A. E.J. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar) and including CFTR N.A. R.J. Am. J. Physiol. PubMed Google Scholar, J. Frizzell R.A. Clin. Am. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, J. M. J. M. M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). In addition to receptor the of basolateral adenosine stimulation with the (10Strohmeier G.R. Reppert S.M. Lencer W.I. Madara J.L. J. Biol. Chem. 1995; 270: 2387-2394Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar) may also be by the of A2bR and its signaling complex in to the in the apical membrane. via apical adenosine receptor may at receptor via coupling to adenylate cyclase, protein kinase and conductance Such efficiency may the between the of this (10Strohmeier G.R. Reppert S.M. Lencer W.I. Madara J.L. J. Biol. Chem. 1995; 270: 2387-2394Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar). data in apical membrane are in with this (19Huang P. Lazarowski E.R. Tarran R. Milgram S.L. Boucher R.C. Stutts M.J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 14120-14125Crossref PubMed Scopus (178) Google Scholar). Our structural evidence that the A2bR upon apical or basolateral agonist stimulation is present in a multiprotein We protein-protein between A2bR and E3KARP and ezrin and E3KARP and PKA with data the recruitment of the A2bR to the plasma membrane these data suggest that the A2bR may be in the plasma membrane by The through of may PKA in to adenylate and Ezrin on the one interacts with actin cytoskeleton and on the as a PKA anchoring protein it in to the receptor signaling complex (21Sun F. Hug M.J. Lewarchik C.M. Yun C.H. Bradbury N.A. Frizzell R.A. J. Biol. Chem. 2000; 275: 29539-29546Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar, C.H. G. A. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). interaction between and the G protein-coupled receptor has been for the receptor (22Hall R.A. Premont R.T. Chow C.W. Blitzer J.T. Pitcher J.A. Claing A. Stoffel R.H. Barak L.S. Shenolikar S. Weinman E.J. Grinstein S. Lefkowitz R.J. Nature. 1998; 392: 626-630Crossref PubMed Scopus (524) Google Scholar). We are the mechanism by which A2bR interacts with In the of the the interaction between the receptor and shown to be by the (S/T)XL motif in the C terminus of the receptor that interacts with (22Hall R.A. Premont R.T. Chow C.W. Blitzer J.T. Pitcher J.A. Claing A. Stoffel R.H. Barak L.S. Shenolikar S. Weinman E.J. Grinstein S. Lefkowitz R.J. Nature. 1998; 392: 626-630Crossref PubMed Scopus (524) Google Scholar). The also that such an interaction not for the coupling of the receptor to but for the of the (22Hall R.A. Premont R.T. Chow C.W. Blitzer J.T. Pitcher J.A. Claing A. Stoffel R.H. Barak L.S. Shenolikar S. Weinman E.J. Grinstein S. Lefkowitz R.J. Nature. 1998; 392: 626-630Crossref PubMed Scopus (524) Google Scholar). Although the A2bR not the motif in the C terminus the is present in the Such interaction between and the motif in has been for interaction C.H. G. A. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). or not the interaction of A2b receptor with and PKA is for coupling or chloride secretion to be Our data that the to adenosine stimulation is in Caco2-BBE cells transfected with a A2bR a in the PDZ motif in the extracellular V. Our data that the A2bR interacts with ezrin and the interaction of ezrin with both E3KARP and A2bR is at upon stimulation of the A2bR of ezrin by Furthermore, data suggest that the of ezrin is not of an in but as which by adenylate cyclase, not interaction C.H. G. A. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). Ezrin is present in cells in both active and The of ezrin its to its and the actin The of ezrin is to be to of a A. Chambers D. R. D. Annu. Rev. Biol. 2000; PubMed Scopus Google Scholar). The mechanism of ezrin by adenosine is and to this is the evidence of of ezrin. In we that the A2bR is recruited to the plasma membrane upon agonist stimulation. The in to agonist interacts with and PKA We speculate that such an interaction not only the receptor in the plasma membrane but to stabilize the receptor in a signaling complex in the membrane that the chloride secretory to We for with of and and for critical of the