Protein Kinase A and G Protein-coupled Receptor Kinase Phosphorylation Mediates β-1 Adrenergic Receptor Endocytosis through Different Pathways

Agonist-induced phosphorylation of β-adrenergic receptors (βARs) by G protein-coupled receptor kinases (GRKs) results in their desensitization followed by internalization. Whether protein kinase A (PKA)-mediated phosphorylation of βARs, particularly the β1AR subtype, can also trigger internalization is currently not known. To test this, we cloned the mouse wild type β1AR (WTβ1AR) and created 3 mutants lacking, respectively: the putative PKA phosphorylation sites (PKA–β1AR), the putative GRK phosphorylation sites (GRK–β1AR), and both sets of phosphorylation sites (PKA–/GRK–β1AR). Following agonist stimulation, both PKA–β1AR and GRK–β1AR mutants showed comparable increases in phosphorylation and desensitization. Saturating concentrations of agonist induced only 50% internalization of either mutant compared with wild type, suggesting that both PKA and GRK phosphorylation of the receptor contributed to receptor sequestration in an additive manner. Moreover, in contrast to the WTβ1AR and PKA–β1AR, sequestration of the GRK–β1AR and PKA–/GRK–β1AR was independent of β-arrestin recruitment. Importantly, clathrin inhibitors abolished agonist-dependent internalization for both the WTβ1AR and PKA–β1AR, whereas caveolae inhibitors prevented internalization only of the GRK–β1AR mutant. Taken together, these data demonstrate that: 1) PKA-mediated phosphorylation can trigger agonist-induced internalization of the β1AR and 2) the pathway selected for β1AR internalization is primarily determined by the kinase that phosphorylates the receptor, i.e. PKA-mediated phosphorylation directs internalization via a caveolae pathway, whereas GRK-mediated phosphorylation directs it through clathrin-coated pits. Agonist-induced phosphorylation of β-adrenergic receptors (βARs) by G protein-coupled receptor kinases (GRKs) results in their desensitization followed by internalization. Whether protein kinase A (PKA)-mediated phosphorylation of βARs, particularly the β1AR subtype, can also trigger internalization is currently not known. To test this, we cloned the mouse wild type β1AR (WTβ1AR) and created 3 mutants lacking, respectively: the putative PKA phosphorylation sites (PKA–β1AR), the putative GRK phosphorylation sites (GRK–β1AR), and both sets of phosphorylation sites (PKA–/GRK–β1AR). Following agonist stimulation, both PKA–β1AR and GRK–β1AR mutants showed comparable increases in phosphorylation and desensitization. Saturating concentrations of agonist induced only 50% internalization of either mutant compared with wild type, suggesting that both PKA and GRK phosphorylation of the receptor contributed to receptor sequestration in an additive manner. Moreover, in contrast to the WTβ1AR and PKA–β1AR, sequestration of the GRK–β1AR and PKA–/GRK–β1AR was independent of β-arrestin recruitment. Importantly, clathrin inhibitors abolished agonist-dependent internalization for both the WTβ1AR and PKA–β1AR, whereas caveolae inhibitors prevented internalization only of the GRK–β1AR mutant. Taken together, these data demonstrate that: 1) PKA-mediated phosphorylation can trigger agonist-induced internalization of the β1AR and 2) the pathway selected for β1AR internalization is primarily determined by the kinase that phosphorylates the receptor, i.e. PKA-mediated phosphorylation directs internalization via a caveolae pathway, whereas GRK-mediated phosphorylation directs it through clathrin-coated pits. β-Adrenergic receptors (βARs) 1The abbreviations used are: βAR, β-adrenergic receptor; GPCR, guanine nucleotide-binding regulatory protein-coupled receptor; PKA, protein kinase A; PKC, protein kinase C; GRK, G protein-coupled receptor kinase; HEK, human embryonal kidney; IBMX, 3-isobutyl-1-methylxanthine; ISO, (–)-isoproterenol bitartrate; β-CD, 2-hydroxypropyl-β-cyclodextrin; MDC, monodansylcadaverine; WTβ1AR, wild type β1AR; PKA–β1AR, β1AR lacking putative PKA phosphorylation sites; GRK–β1AR, β1AR lacking putative GRK phosphorylation sites; PKA–/GRK– β1AR, β1AR lacking both sets of phosphorylation sites; GFP, green fluorescent protein; MEM, minimum essential medium; WT, wild type; Gβγ, beta-gamma subunits of G protein; Gαs, stimulatory G protein alpha subunit; GFX, bisindolylmalemide I; PMA, phorbol 12-myristate 13-acetate; VASP, vasodilator- and A kinase-stimulated phosphoprotein; ELISA, enzyme-linked immunosorbent assay; PBS, phosphate-buffered saline; BSA, bovine serum albumin; Erk, extracellular signal-regulated kinase; ANOVA, analysis of variance. belong to the large family of G protein-coupled receptors (GPCRs) characterized by a typical structure of seven transmembrane domains (1Gudermann T. Nurnberg B. Schultz G. J. Mol. Med. 1995; 73: 51-63Crossref PubMed Scopus (178) Google Scholar, 2Rockman H.A. Koch W.J. Lefkowitz R.J. Nature. 2002; 415: 206-212Crossref PubMed Scopus (788) Google Scholar). Three types of βARs, designated β1, β2, and β3ARs, have been cloned from mammalian tissues (1Gudermann T. Nurnberg B. Schultz G. J. Mol. Med. 1995; 73: 51-63Crossref PubMed Scopus (178) Google Scholar, 3Dohlman H.G. Thorner J. Caron M.G. Lefkowitz R.J. Annu. Rev. Biochem. 1991; 60: 653-688Crossref PubMed Scopus (1137) Google Scholar). Both β1 and β2ARs contain phosphorylation sites located in the third intracellular loop and the C-terminal tail of the receptor, which serve as targets for cAMP-dependent protein kinase A (PKA), protein kinase C (PKC), and G protein-coupled receptor kinases (GRKs) (2Rockman H.A. Koch W.J. Lefkowitz R.J. Nature. 2002; 415: 206-212Crossref PubMed Scopus (788) Google Scholar). Furthermore, site-specific mutagenesis studies of the human β2AR suggest that low concentrations of agonist preferentially induce phosphorylation at PKA sites, whereas higher concentrations of agonist induce phosphorylation at both PKA and GRK sites (4Hausdorff W.P. Bouvier M. O'Dowd B.F. Irons G.P. Caron M.G. Lefkowitz R.J. J. Biol. Chem. 1989; 264: 12657-12665Abstract Full Text PDF PubMed Google Scholar). Continuous exposure of cells to a stimulus causes βARs to undergo rapid phosphorylation in a process that dampens receptor signaling known as desensitization (4Hausdorff W.P. Bouvier M. O'Dowd B.F. Irons G.P. Caron M.G. Lefkowitz R.J. J. Biol. Chem. 1989; 264: 12657-12665Abstract Full Text PDF PubMed Google Scholar, 5Lefkowitz R.J. J. Biol. Chem. 1998; 273: 18677-18680Abstract Full Text Full Text PDF PubMed Scopus (908) Google Scholar, 6Lohse M.J. Benovic J.L. Codina J. Caron M.G. Lefkowitz R.J. Science. 1990; 248: 1547-1550Crossref PubMed Scopus (919) Google Scholar, 7Roth N.S. Campbell P.T. Caron M.G. Lefkowitz R.J. Lohse M.J. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 6201-6204Crossref PubMed Scopus (118) Google Scholar, 8Pitcher J.A. Freedman N.J. Lefkowitz R.J. Ann. Rev. Biochem. 1998; 67: 653-692Crossref PubMed Scopus (1072) Google Scholar). βARs demonstrate two different mechanisms of desensitization. Agonist-specific or homologous desensitization of βARs consists of a two-step process in which phosphorylation at the C terminus of the βAR is mediated by GRKs followed by binding to an arrestin protein, which sterically interrupts signaling to the G protein (5Lefkowitz R.J. J. Biol. Chem. 1998; 273: 18677-18680Abstract Full Text Full Text PDF PubMed Scopus (908) Google Scholar, 8Pitcher J.A. Freedman N.J. Lefkowitz R.J. Ann. Rev. Biochem. 1998; 67: 653-692Crossref PubMed Scopus (1072) Google Scholar). Heterologous or non-agonist-specific desensitization is mediated by the second messenger-stimulated protein kinases A and C, which phosphorylate the receptor and effect a change in receptor conformation such that interaction with the G protein is impaired (5Lefkowitz R.J. J. Biol. Chem. 1998; 273: 18677-18680Abstract Full Text Full Text PDF PubMed Scopus (908) Google Scholar). An important consequence of agonist-mediated receptor phosphorylation and desensitization by GRKs is the subsequent internalization of phosphorylated receptors into the cell (9Claing A. Laporte S.A. Caron M.G. Lefkowitz R.J. Prog. Neurobiol. 2002; 66: 61-79Crossref PubMed Scopus (454) Google Scholar). This process is mediated by β-arrestin, which binds to components of the clathrin-mediated endocytic machinery and targets the ligand-bound receptor to clathrin-coated pits for endocytosis (10Lin F. Wang H. Malbon C.C. J. Biol. Chem. 2000; 275: 19025-19034Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 11Luttrell L.M. Ferguson S.S. Daaka Y. Miller W.E. Maudsley S. Della Rocca G.J. Lin F. Kawakatsu H. Owada K. Luttrell D.K. Caron M.G. Lefkowitz R.J. Science. 1999; 283: 655-661Crossref PubMed Scopus (1264) Google Scholar). Interestingly, PKA phosphorylation, although an important mechanism for desensitization (4Hausdorff W.P. Bouvier M. O'Dowd B.F. Irons G.P. Caron M.G. Lefkowitz R.J. J. Biol. Chem. 1989; 264: 12657-12665Abstract Full Text PDF PubMed Google Scholar, 5Lefkowitz R.J. J. Biol. Chem. 1998; 273: 18677-18680Abstract Full Text Full Text PDF PubMed Scopus (908) Google Scholar, 6Lohse M.J. Benovic J.L. Codina J. Caron M.G. Lefkowitz R.J. Science. 1990; 248: 1547-1550Crossref PubMed Scopus (919) Google Scholar, 7Roth N.S. Campbell P.T. Caron M.G. Lefkowitz R.J. Lohse M.J. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 6201-6204Crossref PubMed Scopus (118) Google Scholar, 8Pitcher J.A. Freedman N.J. Lefkowitz R.J. Ann. Rev. Biochem. 1998; 67: 653-692Crossref PubMed Scopus (1072) Google Scholar), appears to play only a small role in β2AR internalization (12Ferguson S.S. Menard L. Barak L.S. Koch W.J. Colapietro A.M. Caron M.G. J. Biol. Chem. 1995; 270: 24782-24789Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar). Although mechanisms of phosphorylation, desensitization, and internalization by GRKs have been well studied for the β2AR (4Hausdorff W.P. Bouvier M. O'Dowd B.F. Irons G.P. Caron M.G. Lefkowitz R.J. J. Biol. Chem. 1989; 264: 12657-12665Abstract Full Text PDF PubMed Google Scholar, 5Lefkowitz R.J. J. Biol. Chem. 1998; 273: 18677-18680Abstract Full Text Full Text PDF PubMed Scopus (908) Google Scholar, 6Lohse M.J. Benovic J.L. Codina J. Caron M.G. Lefkowitz R.J. Science. 1990; 248: 1547-1550Crossref PubMed Scopus (919) Google Scholar, 7Roth N.S. Campbell P.T. Caron M.G. Lefkowitz R.J. Lohse M.J. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 6201-6204Crossref PubMed Scopus (118) Google Scholar, 8Pitcher J.A. Freedman N.J. Lefkowitz R.J. Ann. Rev. Biochem. 1998; 67: 653-692Crossref PubMed Scopus (1072) Google Scholar), little is known of the role that PKA-mediated phosphorylation plays in the internalization of βARs, particularly the β1AR. GPCRs can internalize via at least two distinct pathways, namely clathrin-coated pits and caveolae. Although very different structurally, clathrin-coated pits and caveolae both serve as microdomains, which, in addition to functioning as transport machinery, also serve as platforms for integrating the cell's signal-transduction pathways (13Anderson R.G. Annu. Rev. Biochem. 1998; 67: 199-225Crossref PubMed Scopus (1727) Google Scholar, 14Brodsky F.M. Chen C.Y. Annu. Rev. Biol. PubMed Scopus Google Scholar). domains serve to different from a signaling pathway these distinct J. Science. PubMed Scopus Google Scholar). to to have located in the of their tail or to contain a binding for a protein that directs the to a R.G. K. Science. 2002; PubMed Scopus Google Scholar, Lefkowitz R.J. 2002; PubMed Scopus Google Scholar). although a of studies have a role for GRK phosphorylation and β-arrestin binding in the process of clathrin-mediated internalization of the β2AR (5Lefkowitz R.J. J. Biol. Chem. 1998; 273: 18677-18680Abstract Full Text Full Text PDF PubMed Scopus (908) Google Scholar), the mechanisms that in the internalization of the β1AR known. of the was to the role of and phosphorylation of the β1AR in the pathway for receptor internalization. cell from embryonal cells from was from and bisindolylmalemide was from the (–)-isoproterenol and phorbol 12-myristate from β2AR was from and from from A and from An and protein from was from mouse wild type β1AR (WTβ1AR) as Barak L.S. A. Caron M.G. H.A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). Three different mutants lacking, respectively: the putative PKA phosphorylation sites (PKA–β1AR), the putative GRK phosphorylation sites (GRK–β1AR), and both sets of sites a of for and into mammalian and cells as Barak L.S. A. Caron M.G. H.A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). cells cells by of of the mutant β1AR or of the WTβ1AR, with the the of β1AR with a into as for phosphorylation and 3 for and studies cells with WTβ1AR, PKA–β1AR, and to cell cells selected by the addition of at a of of receptor was determined by binding WTβ1AR PKA–β1AR GRK–β1AR PKA–/GRK–β1AR of cells used in and cell phosphorylation was as N.J. G. Caron M.G. Lefkowitz R.J. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar). at in was for in of PKA the followed by with for in the β1AR of cell of cell was determined a of cell by of β1AR from and by in to and with an was by binding studies at for 3 as Barak L.S. A. Caron M.G. H.A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). was by with and in a was with a protein with bovine serum as the sequestration a exposure to agonist was as the of binding by by agonist for internalization of the wild type and the receptor was by the as L. J. F. Mol. PubMed Scopus Google Scholar, J. S. Daaka Y. PubMed Scopus Google Scholar). cell the WTβ1AR or the or mutants To of cells to the with in cells with minimum essential for at concentrations of for at by the followed by cells in for at with followed by with in for was at a of for followed by subsequent with with at a of in for and with the addition of a change was for at a cells studied to and in in was in a as Laporte S.A. J.A. Barak L.S. Caron M.G. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). either the WTβ1AR or of the mutants and with in with for a of a and at was in the in which agonist-dependent was and as of the green fluorescent from the to cells for in to the sets of was used as and the for desensitization used for desensitization to for at and with cells in an manner. Both sets of cells with MEM, IBMX, and to for at by of the and addition of of with to at for subsequent of and for and at for and was to a for was the in the of protein was determined a protein was as Barak L.S. A. Caron M.G. H.A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). cells with the either the or of the mutant as well as cells in the or of β-CD, with and for the and with for at of receptor was as Laporte S.A. Caron M.G. Barak L. H.A. J. Biol. 2002; PubMed Scopus Google Scholar). was as Mol. Biol. 2002; PubMed Scopus (178) Google Scholar). the of and of for and and of cells with inhibitors and with agonist at in serum as in the stimulation, cells with and by of was by protein a of protein kinase with as and A kinase-stimulated was by with as in for Agonist-induced of the in the role of and GRK-mediated phosphorylation of the β1AR in an agonist-dependent we used cells with the WTβ1AR, PKA–β1AR, GRK–β1AR, or PKA–/GRK–β1AR the WTβ1AR as a with a of stimulation, phosphorylation of the WTβ1AR as well as PKA–β1AR and GRK–β1AR mutants both the PKA and GRK sites agonist-dependent phosphorylation of the receptor was Agonist-induced of the in the role of PKA and GRK phosphorylation in the desensitization of the β1AR we in cells with the β1AR PKA and GRK phosphorylation mutants a to in to the wild type receptor, that to of the PKA–/GRK–β1AR mutant only a in suggesting that of of the sites in a of receptor Furthermore, cells the WTβ1AR, PKA–β1AR, or GRK–β1AR showed desensitization as a in exposure to the agonist contrast to the wild type receptor, which at agonist concentrations that a in intracellular cells with the PKA–/GRK–β1AR not by with with the β2AR of PKA-mediated desensitization and GRK-mediated homologous desensitization, these data suggest that by either phosphorylation of PKA GRK sites and only the phosphorylation sites a in agonist desensitization Both PKA and GRKs Agonist-induced to desensitization, agonist-induced internalization of β2ARs is to mediated through GRK phosphorylation and β-arrestin To the of PKA and GRK phosphorylation to agonist β1AR we the of from the cell in to by in agonist in a of from the cell at agonist concentrations the of agonist-induced sequestration of both the PKA–β1AR and GRK–β1AR was that of the mutant agonist-induced internalization. results contrast with the desensitization data which that either PKA or GRK sites for receptor desensitization, and suggest that both PKA and GRK phosphorylation for internalization of the β1AR. To the role of PKA and GRK phosphorylation in β1AR receptor we determined the of sequestration of the wild type and mutant receptors WTβ1AR and PKA–β1AR internalization at agonist the for GRK–β1AR internalization was higher for WTβ1AR PKA–β1AR GRK–β1AR and PKA–/GRK–β1AR two distinct mechanisms to to β1AR a GRK mechanism that at agonist and the a PKA mechanism that for of the agonist-dependent β1AR sequestration at higher agonist Importantly, the of sequestration for the β1AR mutants was a of agonist concentrations PKA-mediated to the studies have that GRK-mediated desensitization of β-arrestin to the phosphorylated β2AR (5Lefkowitz R.J. J. Biol. Chem. 1998; 273: 18677-18680Abstract Full Text Full Text PDF PubMed Scopus (908) Google Scholar). studied the of the wild type and mutant receptors to to the in agonist of the WTβ1AR in of β-arrestin from the to the the of PKA sites (PKA–β1AR), β-arrestin was cells the GRK–β1AR mutant showed of β-arrestin to the A and of phosphorylation sites in the mutant in very low β-arrestin comparable to that of the GRK–β1AR A and Importantly, the of the GRK–β1AR sites to internalize the of β-arrestin that internalization of the β1AR can via of the via been to important for the β2AR to clathrin-coated pits for internalization W.E. Lefkowitz R.J. Biol. PubMed Scopus Google Scholar). the GRK–β1AR mutant showed of β-arrestin compared with the PKA–β1AR we to the of internalization and GRK-mediated phosphorylation of the β1AR. these cells WTβ1AR, PKA–β1AR, GRK–β1AR, or β2AR as a used for the internalization studies by caveolae have been to play a role in both the signaling and internalization of cells with two different caveolae pathway a and β-CD, which causes of the caveolae by J. Biol. 1998; PubMed Scopus Google Scholar). in the of intracellular with of the of receptors in and with not internalization of the WTβ1AR, or the PKA–β1AR and prevented of and the of intracellular for the GRK–β1AR mutant sites β-CD, of we only internalization of the GRK–β1AR mutant was Importantly, addition of to the cells with was to the effect the GRK–β1AR mutant agonist of caveolae pathway in cells by internalization of the β1AR. from was used to cells with the that for WTβ1AR, β1AR, and for 3 and in the or of for a of was for with Following with for cells and with cells in and of βAR at the Following agonist stimulation, βARs into with of and with caveolae and internalization of the GRK–β1AR sites of to the cells with the effect the GRK–β1AR mutant and internalization agonist by WTβ1AR, and PKA–β1AR effect internalization and by effect cells not and not PKA cells and with for in and and in the or of the caveolae inhibitors and for and with for agonist stimulation, cell was followed by of cell with J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, J. T. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). PKA phosphorylates and is as a from to by J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, J. T. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). PKA-mediated phosphorylation of was by with the PKA for caveolae contain of the in as signaling as well as for receptor internalization. to the caveolae inhibitors and internalization of the GRK–β1AR mutant by PKA we used a to the of protein kinases in to and A kinase-stimulated is a of protein kinases that an from to phosphorylation J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, J. T. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). in agonist by induced phosphorylation of of cells with either or showed a to that of that the caveolae inhibitors have effect the of of PKA was by of cells to the PKA that PKA and prevented phosphorylation of To test the β1AR a clathrin-coated mechanism for we cells with clathrin with the clathrin inhibitors and S. A.M. Miller W.E. S. Daaka Y. Lefkowitz R.J. Luttrell L.M. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar, Barak L.S. Caron M.G. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google in of internalization for and and an effect internalization of the GRK–β1AR mutant To the of and to clathrin-mediated we their effect internalization of a receptor that was to internalize via clathrin-coated Mol. 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Taken these data demonstrate that PKA-mediated phosphorylation of the β1AR directs internalization via a caveolae pathway, whereas GRK-mediated phosphorylation of the β1AR directs internalization through the clathrin-coated a in phosphorylation directs β1AR internalization through a mechanism of desensitization, cells either the WTβ1AR, PKA–β1AR, or GRK–β1AR used in the internalization of or cells with the GFX, followed by agonist in the of intracellular with of and to cells with agonist and of a effect cells the wild type or the phosphorylation mutant receptors that phosphorylation not play a role in β1AR internalization these To the of to phosphorylation we effect cells the WTβ1AR by for with a large in the of the of with the in of or with Taken the results was to in the second that phosphorylation not play a role in β1AR we demonstrate in addition to the role of GRKs in the process of receptor PKA-mediated phosphorylation plays a role in agonist-induced internalization of the β1AR. Furthermore, although GRK-mediated phosphorylation directs internalization through a clathrin-coated pathway, PKA-mediated phosphorylation directs internalization via a caveolae data contrast with PKA and GRK mutants of the Although both PKA and GRK phosphorylation to desensitization of the β2AR (4Hausdorff W.P. Bouvier M. O'Dowd B.F. Irons G.P. Caron M.G. Lefkowitz R.J. J. Biol. Chem. 1989; 264: 12657-12665Abstract Full Text PDF PubMed Google Scholar, S.S. Menard L. Barak L.S. Koch W.J. Colapietro A.M. Caron M.G. J. Biol. Chem. 1995; 270: 24782-24789Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar), PKA phosphorylation not play a role in endocytosis of receptor (12Ferguson S.S. Menard L. Barak L.S. Koch W.J. Colapietro A.M. Caron M.G. J. Biol. 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