Receptor Number and Caveolar Co-localization Determine Receptor Coupling Efficiency to Adenylyl Cyclase

Recent evidence suggests that many signaling molecules localize in microdomains of the plasma membrane, particularly caveolae. In this study, overexpression of adenylyl cyclase was used as a functional probe of G protein-coupled receptor (GPCR) compartmentation. We found that three endogenous receptors in neonatal rat cardiomyocytes couple with different levels of efficiency to the activation of adenylyl cyclase type 6 (AC6), which localizes to caveolin-rich membrane fractions. Overexpression of AC6 enhanced the maximal cAMP response to β1-adrenergic receptor (β1AR)-selective activation 3.7-fold, to β2AR-selective activation only 1.6-fold and to prostaglandin E2 (PGE2) not at all. Therefore, the rank order of efficacy in coupling to AC6 is β1AR > β2AR > prostaglandin E2 receptor (EP2R). β2AR coupling efficiency was greater when we overexpressed the receptor or blocked its desensitization by expressing βARKct, an inhibitor of G protein-coupled receptor kinase activation, but was not significantly greater when cells were treated with pertussis toxin. Assessment of receptor and AC expression indicated co-localization of AC5/6, β1AR, and β2AR, but not EP2R, in caveolin-rich membranes and caveolin-3 immunoprecipitates, likely explaining the observed activation of AC6 by βAR subtypes but lack thereof by PGE2. When cardiomyocytes were stimulated with a βAR agonist, β2AR were no longer found in caveolin-3 immunoprecipitates; an effect that was blocked by expression of βARKct. Thus, agonist-induced translocation of β2AR out of caveolae causes a sequestration of receptor from effector and likely contributes to the lower efficacy of β2AR coupling to AC6 as compared with β1AR, which do not similarly translocate. Therefore, spatial co-localization is a key determinant of efficiency of coupling by particular extracellular signals to activation of GPCR-linked effectors. Recent evidence suggests that many signaling molecules localize in microdomains of the plasma membrane, particularly caveolae. In this study, overexpression of adenylyl cyclase was used as a functional probe of G protein-coupled receptor (GPCR) compartmentation. We found that three endogenous receptors in neonatal rat cardiomyocytes couple with different levels of efficiency to the activation of adenylyl cyclase type 6 (AC6), which localizes to caveolin-rich membrane fractions. Overexpression of AC6 enhanced the maximal cAMP response to β1-adrenergic receptor (β1AR)-selective activation 3.7-fold, to β2AR-selective activation only 1.6-fold and to prostaglandin E2 (PGE2) not at all. Therefore, the rank order of efficacy in coupling to AC6 is β1AR > β2AR > prostaglandin E2 receptor (EP2R). β2AR coupling efficiency was greater when we overexpressed the receptor or blocked its desensitization by expressing βARKct, an inhibitor of G protein-coupled receptor kinase activation, but was not significantly greater when cells were treated with pertussis toxin. Assessment of receptor and AC expression indicated co-localization of AC5/6, β1AR, and β2AR, but not EP2R, in caveolin-rich membranes and caveolin-3 immunoprecipitates, likely explaining the observed activation of AC6 by βAR subtypes but lack thereof by PGE2. When cardiomyocytes were stimulated with a βAR agonist, β2AR were no longer found in caveolin-3 immunoprecipitates; an effect that was blocked by expression of βARKct. Thus, agonist-induced translocation of β2AR out of caveolae causes a sequestration of receptor from effector and likely contributes to the lower efficacy of β2AR coupling to AC6 as compared with β1AR, which do not similarly translocate. Therefore, spatial co-localization is a key determinant of efficiency of coupling by particular extracellular signals to activation of GPCR-linked effectors. G protein-coupled receptor β-adrenergic receptor adenylyl cyclase isoproterenol prostaglandin E2, EP2R, prostanoid EP2 receptor G protein receptor kinase β-adrenergic receptor kinase C-terminal peptide of βARK multiplicity of infection 4-morpholineethanesulfonic acid cAMP-dependent kinase protein kinase C phosphate-buffered saline Regulation of cells by G protein-coupled receptors (GPCRs)1 involves the sequential flow of information from the receptors via heterotrimeric G proteins to effector molecules. Among the GPCRs, probably the most well studied are the β-adrenergic receptors (βAR), which are widely expressed in cells throughout the body, including cardiac myocytes. Cardiac βAR, which are activated by neuronally released and circulating catecholamines, change several aspects of cardiac function, including increases in the rate and force of contraction and the rate of relaxation. βAR exerts the majority of its effects by coupling to the heterotrimeric G protein, Gs, and the stimulation of adenylyl cyclase (AC) activity. Activity of AC produces the second messenger cAMP, which, via activation of protein kinase A, alters intracellular Ca2+ dynamics and contractile function by phosphorylating calcium channels, troponin I, and phospholamban (1Rapundalo S.T. Solaro R.J. Kranias E.G. Circ. Res. 1989; 64: 104-111Crossref PubMed Scopus (115) Google Scholar, 2Hartzell H.C. Mery P.F. Fischmeister R. Szabo G. Nature. 1991; 351: 573-576Crossref PubMed Scopus (188) Google Scholar). The β1AR is the predominant subtype in cardiac myocytes, outnumbering β2AR by ∼4:1 (3Kuznetsov V. Pak E. Robinson R.B. Steinberg S.F. Circ. Res. 1995; 76: 40-52Crossref PubMed Google Scholar, 4Morisco C. Zebrowski D.C. Vatner D.E. Vatner S.F. Sadoshima J. J. Mol. Cell Cardiol. 2001; 33: 561-573Abstract Full Text PDF PubMed Scopus (121) Google Scholar). However, interest has grown recently in the possibility that these receptor subtypes are not redundant, but differentially regulate cardiac function. The distinct physiological actions of β1AR and β2AR may represent coupling to different signaling pathways (5Xiao R.P. Lakatta E.G. Circ. Res. 1993; 73: 286-300Crossref PubMed Scopus (209) Google Scholar, 6Xiao R.P. Hohl C. Altschuld R. Jones L. Livingston B. Ziman B. Tantini B. Lakatta E.G. J. Biol. Chem. 1994; 269: 19151-19156Abstract Full Text PDF PubMed Google Scholar, 7Xiao R.P. Avdonin P. Zhou Y.Y. Cheng H. Akhter S.A. Eschenhagen T. Lefkowitz R.J. Koch W.J. Lakatta E.G. Circ. Res. 1999; 84: 43-52Crossref PubMed Scopus (335) Google Scholar, 8Yatani A. Tajima Y. Green S.A. Cell Signal. 1999; 11: 337-342Crossref PubMed Scopus (21) Google Scholar) and/or different spatial localization within the heart or within single cells (9Steinberg S.F. Circ. Res. 1999; 85: 1101-1111Crossref PubMed Scopus (206) Google Scholar, 10Rybin V.O. Xu X. Lisanti M.P. Steinberg S.F. J. Biol. Chem. 2000; 275: 41447-41457Abstract Full Text Full Text PDF PubMed Scopus (465) Google Scholar). Expression of both βAR and AC in distinct caveolar microdomains of the plasma membrane has been recently demonstrated in both non-cardiac and cardiac cells (10Rybin V.O. Xu X. Lisanti M.P. Steinberg S.F. J. Biol. Chem. 2000; 275: 41447-41457Abstract Full Text Full Text PDF PubMed Scopus (465) Google Scholar, 11Schwencke C. Yamamoto M. Okumura S. Toya Y. Kim S.J. Ishikawa Y. Mol. Endocrinol. 1999; 13: 1061-1070PubMed Google Scholar, 12Schwencke C. Okumura S. Yamamoto M. Geng Y.J. Ishikawa Y. J. Cell. Biochem. 1999; 75: 64-72Crossref PubMed Scopus (98) Google Scholar, 13Ostrom R.S. Violin J.D. Coleman S. Insel P.A. Mol. Pharmacol. 2000; 57: 1075-1079PubMed Google Scholar, 14Davare M.A. Avdonin V. Hall D.D. Peden E.M. Burette A. Weinberg R.J. Horne M.C. Hoshi T. Hell J.W. Science. 2001; 293: 98-101Crossref PubMed Scopus (441) Google Scholar). Caveolae, detectable as plasma membrane invaginations, are enriched in the protein caveolin and in sphingolipid and cholesterol, thereby representing a distinct protein and lipid environment, which appears to attract and retain a subset of plasma membrane proteins (15Shaul P.W. Smart E.J. Robinson L.J. German Z. Yuhanna I.S. Ying Y. Anderson R.G. Michel T. J. Biol. Chem. 1996; 271: 6518-6522Abstract Full Text Full Text PDF PubMed Scopus (626) Google Scholar, 16Anderson R. Annu. Rev. Biochem. 1998; 67: 199-225Crossref PubMed Scopus (1725) Google Scholar). Compartmentation of βAR and AC (and portions of cellular Gs) challenges the concept that components of GPCR signal transduction are randomly distributed protein entities that interact via diffusion over long distances in the plasma membrane. Instead, the sparsely expressed signaling proteins may be restricted to such plasmalemmal microdomains to facilitate rapid and specific signal transduction (17Ostrom R.S. Post S.R. Insel P.A. J. Pharmacol. Exp. Ther. 2000; 294: 407-412PubMed Google Scholar, 18Steinberg S.F. Brunton L.L. Annu. Rev. Pharmacol. Toxicol. 2001; 41: 751-773Crossref PubMed Scopus (320) Google Scholar) and regulation by co-localized molecules (19Fagan K.A. Smith K.E. Cooper D.M. J. Biol. Chem. 2000; 275: 26530-26537Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). We previously reported that overexpression of AC6 in neonatal rat cardiac myocytes selectively enhances responses to βAR activation but not to activation of prostanoid, adenosine, glucagon, or histamine receptors and found that AC6 and β1AR are co-localized in caveolar membranes of cardiomyocytes (13Ostrom R.S. Violin J.D. Coleman S. Insel P.A. Mol. Pharmacol. 2000; 57: 1075-1079PubMed Google Scholar). The goal of the present study was to test the role of receptor-effector co-localization in caveolae as a contributor to the ability of different GPCRs to regulate second messenger generation. We used AC6 overexpression as a read-out of receptor coupling by comparing the degree to which it enhanced maximal cAMP response to receptor activation. We show here that β1AR couple more efficaciously to AC6 as compared with β2AR, but that increasing total expression of β2AR or blocking their desensitization and translocation out of caveolae enhances the coupling of this receptor to AC6. Taken together with the fact that prostanoid receptors, which do not couple to AC6, were not found in caveolae, we conclude that GPCR ability to couple to the stimulation of AC6 depends upon the co-localization of sufficient active receptors in caveolin-rich membrane microdomains, the predominant subcellular site of AC6 expression. Adenovirus-expressing murine AC6 was generated as described previously (20Gao M. Ping P. Post S. Insel P.A. Tang R. Hammond H.K. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 1038-1043Crossref PubMed Scopus (108) Google Scholar). βARKct adenovirus was a gift from Robert Lefkowitz (Duke University). Primary antibodies for β1AR, β2AR, and AC5/6 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Primary antibody for caveolin-3 was obtained from BD PharMingen. Radiolabeled chemicals were obtained from PerkinElmer Life Sciences. All other chemicals and reagents were obtained from Sigma Chemical. Neonatal cardiac myocytes were prepared and maintained as described previously (13Ostrom R.S. Violin J.D. Coleman S. Insel P.A. Mol. Pharmacol. 2000; 57: 1075-1079PubMed Google Scholar, 20Gao M. Ping P. Post S. Insel P.A. Tang R. Hammond H.K. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 1038-1043Crossref PubMed Scopus (108) Google Scholar). Cells were infected 2 days after plating with indicated adenoviral construct(s) for 20 h (10–100 MOI/cell). Control cells were treated with an identical adenoviral construct containing the lacZ gene. After infection, cells were washed extensively and allowed to equilibrate for 24 h. Membranes were prepared by rinsing cells twice in cells 2 and with 20 in a was at for at was to a and at for The was in membrane 2 to total protein In other AC was in caveolin-3 Cells were and in a with a lower of 2 inhibitor was with of protein for at with caveolin-3 antibody for h. were with protein and in membrane The was by of or containing and and of The was for at and were by for cAMP of was for cAMP by as described previously (13Ostrom R.S. Violin J.D. Coleman S. Insel P.A. Mol. Pharmacol. 2000; 57: 1075-1079PubMed Google Scholar). protein was a protein Neonatal cardiac myocytes were prepared and treated as described and were washed three with and with 20 Cells were for and for cAMP by with of interest in the of for When were these were with cells for of was and of acid was to well to were for cAMP by Neonatal rat cardiac myocytes were a from S. T. M. Lisanti M.P. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar). cells were washed twice in and the in a total of 2 of Cells were with a with three and a with three The was to by of an of in and in an was of the by of prepared in with of in The was at a for h at The at the was as the caveolin-rich and the lower was as the In were to isoproterenol for washed in and were a protein of neonatal rat cardiac myocytes were washed twice with in of inhibitor and with in a were with protein with antibody for h a at were by with protein a at and at for were washed in by in 2 were in containing and at for in the were by in or were by and to a membrane by In protein from were with acid by with acid for at for acid was and were washed with and in for Membranes were blocked in 20 with and with antibody at antibodies were antibody with (Santa Cruz and All at the as by to (Santa Cruz The of protein was a protein that overexpression of AC6 an adenovirus expression in neonatal rat cardiac myocytes selectively enhances βAR responses compared with several GPCR that are efficaciously to and the stimulation of cAMP (13Ostrom R.S. Violin J.D. Coleman S. Insel P.A. Mol. Pharmacol. 2000; 57: 1075-1079PubMed Google Scholar). out the possibility that the intracellular to this we AC in membranes from neonatal rat cardiac myocytes. Membranes from cells or from cells AC6 were maximal of or and responses were greater in membranes from cells In which stimulated AC over was not different in membranes from or Therefore, AC6 overexpression selectively enhances activation of AC but not response to PGE2. responses in cardiac myocytes from activation of different receptors, β1AR and We to the effects of AC6 overexpression responses to the subtypes of βAR (and by receptor coupling to by and responses to of to an of and a of of protein in cells and an of a of of cAMP in cells AC6 2 and with that increasing expression of AC6 enhances maximal response (13Ostrom R.S. Violin J.D. Coleman S. Insel P.A. Mol. Pharmacol. 2000; 57: 1075-1079PubMed Google Scholar, 20Gao M. Ping P. Post S. Insel P.A. Tang R. Hammond H.K. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 1038-1043Crossref PubMed Scopus (108) Google Scholar). However, the of AC overexpression was different β1AR and the maximal response to in the of was enhanced in cells as compared with 2 In the maximal response to in the of was enhanced only 1.6-fold 2 with their actions as both and for in and cells Therefore, the show that AC6 overexpression enhances response more and for both receptors maximal response was of AC and/or receptor expression the and efficacy of isoproterenol in cAMP and overexpression expression and of of of stimulation of cAMP in the of and/or β2AR-selective and from and are in cardiomyocytes expressing β2AR or βARKct in to or AC6. in a and of of of stimulation of cAMP in the of and/or β2AR-selective and from and are in cardiomyocytes expressing β2AR or βARKct in to or AC6. Recent that β2AR may couple to both and R.P. Avdonin P. Zhou Y.Y. Cheng H. Akhter S.A. Eschenhagen T. Lefkowitz R.J. Koch W.J. Lakatta E.G. Circ. Res. 1999; 84: 43-52Crossref PubMed Scopus (335) Google Scholar, R.P. X. Lakatta E.G. Mol. Pharmacol. 1995; PubMed Scopus Google Scholar). coupling of this receptor to its efficacy in we cAMP responses to a maximal of in the or of or in cells treated with pertussis for no effect the of by AC6 overexpression to or β1AR response not in AC6 overexpression a greater effect the β2AR-selective response in cells effect not levels of by and not the response to levels by β1AR activation. levels of cAMP response to were enhanced by of cells with pertussis not to a role cAMP in response to βAR activation. the total βAR in cardiac myocytes is of β1AR and β2AR (3Kuznetsov V. Pak E. Robinson R.B. Steinberg S.F. Circ. Res. 1995; 76: 40-52Crossref PubMed Google we to the lack of effect of AC6 overexpression β2AR responses be to lower expression of this receptor We used an adenovirus to an β2AR in cardiomyocytes such that the of β2AR expression was endogenous levels not cAMP that β2AR in isoproterenol a to the in and in maximal response as compared with cells and AC6 in to β2AR in a maximal response that was greater as compared with cells only Therefore, overexpression of β2AR enhances its ability to couple to AC6 and to cAMP via AC6. has been recently reported that β2AR, but not β1AR, out of caveolin-rich membrane from neonatal rat cardiac myocytes upon stimulation (10Rybin V.O. Xu X. Lisanti M.P. Steinberg S.F. J. Biol. Chem. 2000; 275: 41447-41457Abstract Full Text Full Text PDF PubMed Scopus (465) Google Scholar). We that this translocation be with βARK desensitization of β2AR and thereby to the lower ability of this receptor to couple to the stimulation of AC6. test this we used an adenovirus to the C-terminal peptide of βARK which activation of endogenous βARK by S. Koch W.J. Lefkowitz R.J. J. PubMed Scopus Google thereby desensitization of βAR (and other and increasing signaling W.J. P. Lefkowitz R.J. Science. 1995; PubMed Scopus Google Scholar). We found that activation of β2AR in cardiomyocytes to βARKct a in a in maximal cAMP as compared with and In this of functional β2AR, overexpression of AC6 the maximal response to in cells that only expressed βARKct. that of βAR desensitization greater coupling of β2AR to AC6. In βARKct expression was not to coupling of receptors to overexpressed AC6, that the effect of βARKct was specific to βAR receptors, and not to or AC activation βARKct not cAMP in of cardiomyocytes of caveolin-rich membrane from cardiomyocytes a has indicated that AC5/6 is expressed in caveolin-rich (10Rybin V.O. Xu X. Lisanti M.P. Steinberg S.F. J. Biol. Chem. 2000; 275: 41447-41457Abstract Full Text Full Text PDF PubMed Scopus (465) Google R.S. Violin J.D. Coleman S. Insel P.A. Mol. Pharmacol. 2000; 57: 1075-1079PubMed Google Scholar). Overexpression of AC6 increases AC5/6 in this and 13Ostrom R.S. Violin J.D. Coleman S. Insel P.A. Mol. Pharmacol. 2000; 57: 1075-1079PubMed Google Scholar). In β1AR and expressed were not in EP2 receptor was detectable only in an for the of receptor coupling to overexpressed AC6 the of an effect EP2 receptor responses AC6 overexpression with the lack of co-localization of this receptor and AC6. an we and the proteins that and by AC activity. AC in caveolin-3 as was stimulated by and but not by In AC in the cellular not by caveolin-3 be stimulated by PGE2. AC was in the caveolin-3 in the of the caveolin-3 demonstrated the of β1AR, AC5/6, and expressed but not not caveolin-3 was in both β1AR and AC5/6 from cells as well as in the from cells expressing indicated that β1AR and AC5/6 but that β2AR not with β1AR or AC5/6 not Therefore, β1AR and AC5/6 and and to a β2AR, signaling that (10Rybin V.O. Xu X. Lisanti M.P. Steinberg S.F. J. Biol. Chem. 2000; 275: 41447-41457Abstract Full Text Full Text PDF PubMed Scopus (465) Google Scholar) reported that causes a translocation of β2AR, but not β1AR, out of caveolin-rich from cardiomyocytes (9Steinberg S.F. Circ. Res. 1999; 85: 1101-1111Crossref PubMed Scopus (206) Google Scholar). We cells expressing to for and found that was in caveolin-3 When these were in cardiomyocytes expressing βARKct, in caveolin-3 was and no longer with caveolin-3 stimulation βARKct expression the of β1AR or Therefore, by (9Steinberg S.F. Circ. Res. 1999; 85: 1101-1111Crossref PubMed Scopus (206) Google Scholar) or caveolin-3 β2AR out of caveolae upon translocation appears upon and as for β2AR via L. Science. 1996; 271: PubMed Scopus Google Scholar, R.B. Nature. 1996; PubMed Scopus Google Scholar). We show that βARKct expression this β2AR translocation and enhances β2AR coupling to AC the effect likely from an of functional receptors in the enriched in AC6. is well that cells their and cellular by expressing only a subset of the of receptors that a the extracellular signals to which that However, receptors may signal transduction spatial to particular of receptors with signaling a of in the that receptors and other membrane proteins are randomly distributed in the plasma membrane. The present that caveolin-rich plasma membrane microdomains that are enriched in many signaling receptors and to couple only receptors to a from by and that such caveolin-rich represent caveolae (13Ostrom R.S. Violin J.D. Coleman S. Insel P.A. Mol. Pharmacol. 2000; 57: 1075-1079PubMed Google Scholar, V.O. Xu X. Steinberg S.F. Circ. Res. 1999; 84: PubMed Scopus Google Scholar). as well as regulation of signaling be a determinant for cellular response to extracellular and S.F. Brunton L.L. Annu. Rev. Pharmacol. Toxicol. 2001; 41: 751-773Crossref PubMed Scopus (320) Google Scholar). with we that cardiomyocytes a of signaling in caveolin-rich membranes (10Rybin V.O. Xu X. Lisanti M.P. Steinberg S.F. J. Biol. Chem. 2000; 275: 41447-41457Abstract Full Text Full Text PDF PubMed Scopus (465) Google Scholar, 11Schwencke C. Yamamoto M. Okumura S. Toya Y. Kim S.J. Ishikawa Y. Mol. Endocrinol. 1999; 13: 1061-1070PubMed Google Scholar, 13Ostrom R.S. Violin J.D. Coleman S. Insel P.A. Mol. Pharmacol. 2000; 57: 1075-1079PubMed Google Scholar). We show that type of receptor of the is from caveolin-rich and is to couple to overexpressed AC6. Therefore, localization in this subcellular is a determinant for efficacy of GPCR coupling to an effector β1AR, expressed at and more in caveolae, couple with efficacy to AC6. β2AR, expressed at lower levels as compared with β1AR, in caveolae but out of this upon activation (10Rybin V.O. Xu X. Lisanti M.P. Steinberg S.F. J. Biol. Chem. 2000; 275: 41447-41457Abstract Full Text Full Text PDF PubMed Scopus (465) Google Scholar). translocation within the of cAMP and appears to to the ability of β2AR to AC6. for β1AR, β2AR and of the functional of co-localization of GPCR signaling molecules in caveolin-rich and do endogenous receptors in of a caveolae may as that signaling pathways with other signaling and signaling proteins in and spatial of caveolae been as of Ca2+ M. M. V. M. H. M. Circ. Res. 2000; PubMed Scopus Google Scholar) in cardiomyocytes and the of Ca2+ in other cells (19Fagan K.A. Smith K.E. Cooper D.M. J. Biol. Chem. 2000; 275: 26530-26537Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). Ca2+ is in caveolae, expression of AC6 in this in cAMP levels that are to from the In this is the in which AC6 and caveolae for Ca2+ to AC (19Fagan K.A. Smith K.E. Cooper D.M. J. Biol. Chem. 2000; 275: 26530-26537Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). co-localization in cardiomyocytes to the in cAMP levels observed in heart the cardiac G. Science. PubMed Scopus Google Scholar) and to regulation of cAMP levels by L. L. Smith Michel T. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar). the localization of AC6 expression be to in a different of regulation of cAMP with this AC is when caveolae are with a that from the plasma membrane and of from endogenous AC (10Rybin V.O. Xu X. Lisanti M.P. Steinberg S.F. J. Biol. Chem. 2000; 275: 41447-41457Abstract Full Text Full Text PDF PubMed Scopus (465) Google Scholar). that from is that overexpression of different of AC in different of regulation and different physiological overexpression of AC other that AC6 has been reported to different effects cellular and cardiac Overexpression of which is activated by GPCRs, to a regulation of the of GPCR responses L. G. M.C. J. Circ. Res. 2000; PubMed Scopus Google Scholar). overexpression of in increases in AC and a of responses A. R. Kranias E.G. 1999; PubMed Scopus Google Scholar). with here and with from overexpression of AC6 D.M. Zhou J. J. T. Hammond H.K. 1999; PubMed Scopus Google Scholar). and AC6 are in their regulation by Ca2+ and but is stimulated AC6 is by in regulation may be that to caveolae upon activation V.O. Xu X. Steinberg S.F. Circ. Res. 1999; 84: PubMed Scopus Google Scholar). However, most has not of co-localization of AC and receptors that regulate its activity. The present the of the subcellular localization of both AC and receptor expression. are of the present is that the co-localization of proteins upon a of membrane fractions. may not be caveolae. to a (13Ostrom R.S. Violin J.D. Coleman S. Insel P.A. Mol. Pharmacol. 2000; 57: 1075-1079PubMed Google such may represent different microdomains of the such as lipid P. Mol. Biol. Cell. 2001; PubMed Scopus Google Scholar). the in this were to this of co-localization of receptors and AC likely such as at the antibodies are of in these of B. and P. it is that localization of components in the neonatal may not be identical with in to test this to be In it is to that βARKct activation by S. Koch W.J. Lefkowitz R.J. J. PubMed Scopus Google Scholar). Thus, in cAMP from βARKct expression may be in to the of signaling by and other of In these of signal transduction in cardiomyocytes that of receptors and AC the efficacy of receptor coupling in that different GPCR show different of cAMP which with co-localization in caveolin-rich The co-localization of components in such microdomains has for the regulation of cellular responses to extracellular and that has of signal transduction has and of The here the role of spatial at a subcellular of key signaling molecules in the ability of cells to to extracellular be of interest to such spatial as a of and in The of signaling microdomains in caveolin-rich of cardiomyocytes and other cells may a to for the and of signal We the for their to this H. Hammond for adenovirus with Robert Lefkowitz for adenovirus with H. L. and for with