Retrovirally Mediated Transfer of a G Protein-coupled Receptor Kinase (GRK) Dominant-negative Mutant Enhances Endogenous Calcitonin Receptor Signaling in Chinese Hamster Ovary Cells (2000)

G protein-coupled receptor kinases (GRKs) initiate pathways leading to agonist-dependent phosphorylation and desensitization of G protein-coupled receptors. However, the role of GRKs in modulation of signaling properties of native receptors has not been clearly defined. Here we addressed this question by generating Chinese hamster ovary (CHO) cells stably expressing a dominant-negative mutant of GRK2 (DN-GRK2), K220R, using retrovirally mediated gene transfer, and we assessed function of the endogenously expressed calcitonin (CT) receptors. We found that CT-mediated responses were prominently enhanced in CHO cells expressing DN-GRK2 compared with mock-infected control CHO cells with ∼3-fold increases in CT-promoted cAMP production in whole cells and adenylyl cyclase activity in membrane fractions. CT-promoted phosphoinositide hydrolysis was also enhanced in DN-GRK2 cells. The number of CT receptors was increased ∼3-fold in DN-GRK2 cells, as assessed by125I-salmon CT-specific binding, and this was associated with increased CT receptor mRNA levels. These results indicate that DN-GRK2 has multiple consequences for CT receptor signaling, but a primary effect is an increase in CT receptor mRNA and receptor number and, in turn, enhanced CT receptor signaling. As such, our findings provide a mechanistic basis for previous observations regarding agonist-promoted down-regulation of CT receptors and for resistance and escape from response to CT in vitroand in vivo. Moreover, the data suggest that blunting of receptor desensitization by DN-GRK2 blocks a GRK-mediated tonic inhibition of CT receptor expression and response. We speculate that GRKs play a similar role for other G protein-coupled receptors as well. G protein-coupled receptor kinases (GRKs) initiate pathways leading to agonist-dependent phosphorylation and desensitization of G protein-coupled receptors. However, the role of GRKs in modulation of signaling properties of native receptors has not been clearly defined. Here we addressed this question by generating Chinese hamster ovary (CHO) cells stably expressing a dominant-negative mutant of GRK2 (DN-GRK2), K220R, using retrovirally mediated gene transfer, and we assessed function of the endogenously expressed calcitonin (CT) receptors. We found that CT-mediated responses were prominently enhanced in CHO cells expressing DN-GRK2 compared with mock-infected control CHO cells with ∼3-fold increases in CT-promoted cAMP production in whole cells and adenylyl cyclase activity in membrane fractions. CT-promoted phosphoinositide hydrolysis was also enhanced in DN-GRK2 cells. The number of CT receptors was increased ∼3-fold in DN-GRK2 cells, as assessed by125I-salmon CT-specific binding, and this was associated with increased CT receptor mRNA levels. These results indicate that DN-GRK2 has multiple consequences for CT receptor signaling, but a primary effect is an increase in CT receptor mRNA and receptor number and, in turn, enhanced CT receptor signaling. As such, our findings provide a mechanistic basis for previous observations regarding agonist-promoted down-regulation of CT receptors and for resistance and escape from response to CT in vitroand in vivo. Moreover, the data suggest that blunting of receptor desensitization by DN-GRK2 blocks a GRK-mediated tonic inhibition of CT receptor expression and response. We speculate that GRKs play a similar role for other G protein-coupled receptors as well. G protein-coupled receptors Chinese hamster ovary G protein-coupled receptor kinases calcitonin salmon CT human CT base pair dominant-negative guanosine 5′-3-O-(thio)triphosphate polyacrylamide gel electrophoresis reverse transcriptase-polymerase chain reaction dominant-negative mutant of GRK2 vesicular stomatitis virus multiplicity of infection isobutylmethylxanthine inositol phosphate glyceraldehyde-3-phosphate dehydrogenase confidence intervals calcitonin receptor Stimulation of G protein-coupled receptors (GPCRs)1 in the plasma membrane triggers two events, activation of G protein-mediated signal transduction pathways and, in parallel, a deactivation or desensitization of signaling. One component of this desensitization, in particular homologous, receptor-specific desensitization, is agonist-dependent phosphorylation of the receptor by specific G protein-coupled receptor kinases (GRKs). This phosphorylation event leads to the recruitment of cytosolic proteins, β-arrestins, to the receptor-signaling complex, the uncoupling of receptor from heterotrimeric G proteins, and loss of receptor responsiveness. Recent evidence indicates that GRKs and β-arrestins not only promote receptor uncoupling but also may directly participate in GPCR sequestration and the initiation of events leading to clathrin-coated pit-mediated internalization of receptors (for recent reviews see Refs. 1Pitcher J.A. Freedman N.J. Lefkowitz R.J. Annu. Rev. Biochem. 1998; 67: 653-692Crossref PubMed Scopus (1060) Google Scholar, 2Krupnick J.G. Benovic J.L. Annu. Rev. Pharmacol. Toxicol. 1998; 38: 289-319Crossref PubMed Scopus (855) Google Scholar, 3Lefkowitz R.J. J. Biol. Chem. 1998; 273: 18677-18680Abstract Full Text Full Text PDF PubMed Scopus (903) Google Scholar, 4Hall R.A. Premont R.T. Lefkowitz R.J. J. Cell Biol. 1999; 145: 927-932Crossref PubMed Scopus (279) Google Scholar, 5Bunemann M. Hosey M.M. J. Physiol. ( Lond. ). 1999; 517: 5-23Crossref PubMed Scopus (163) Google Scholar). In addition, GRK activation may be required to initiate certain events unrelated to receptor desensitization (3Lefkowitz R.J. J. Biol. Chem. 1998; 273: 18677-18680Abstract Full Text Full Text PDF PubMed Scopus (903) Google Scholar,4Hall R.A. Premont R.T. Lefkowitz R.J. J. Cell Biol. 1999; 145: 927-932Crossref PubMed Scopus (279) Google Scholar).At least six different isoforms of the GRK family have been isolated. GRK2, formerly termed βARK1, is a widely expressed member of this family and has been shown to phosphorylate various GPCRs (1Pitcher J.A. Freedman N.J. Lefkowitz R.J. Annu. Rev. Biochem. 1998; 67: 653-692Crossref PubMed Scopus (1060) Google Scholar,2Krupnick J.G. Benovic J.L. Annu. Rev. Pharmacol. Toxicol. 1998; 38: 289-319Crossref PubMed Scopus (855) Google Scholar). GRK2 K220R, a dominant-negative GRK2 mutant (DN-GRK2) in which lysine at position 220 has been mutated to arginine to disrupt kinase activity (6Kong G. Penn R. Benovic J.L. J. Biol. Chem. 1994; 269: 13084-13087Abstract Full Text PDF PubMed Google Scholar), has been used to attenuate desensitization of several GPCR systems such as the β2-adrenergic receptor, α1B-adrenergic receptor, adenosine A2 receptor, thyrotropin receptor, follitropin receptor, and CCR2B receptor (6Kong G. Penn R. Benovic J.L. J. Biol. Chem. 1994; 269: 13084-13087Abstract Full Text PDF PubMed Google Scholar, 7Diviani D. Lattion A.L. Larbi N. Kunapuli P. Pronin A. Benovic J.L. Cotecchia S. J. Biol. Chem. 1996; 271: 5049-5058Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar, 8Mundell S.J. Benovic J.L. Kelly E. Mol. Pharmacol. 1997; 51: 991-998Crossref PubMed Scopus (63) Google Scholar, 9Iacovelli L. Franchetti R. Grisolia D. De Blasi A. Mol. Pharmacol. 1999; 56: 316-324Crossref PubMed Scopus (53) Google Scholar, 10Lazari M.F. Liu X. Nakamura K. Benovic J.L. Ascoli M. Mol. Endocrinol. 1999; 13: 866-878Crossref PubMed Scopus (93) Google Scholar, 11Aragay A.M. Mellado M. Frade J.M. Martin A.M. Jimenez-Sainz M.C Martinez- A, C Mayor F. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2985-2990Crossref PubMed Scopus (141) Google Scholar).To date, however, relatively little is known about the role of GRKs in desensitization to peptide hormones, in particular, in the regulation and expression of endogenously expressed GPCRs. Thus, most studies of GRKs have involved the use of transfected cells expressing relatively nonphysiological levels of GPCRs. In the current studies, we utilized a model cell, Chinese hamster ovary (CHO) cells, which endogenously expresses calcitonin (CT) receptors, and we evaluated the role of endogenously expressed GRKs on signaling. We stably expressed DN-GRK2 in CHO cells using retrovirally mediated gene transfer and found that CT receptor expression and signaling were markedly enhanced by the DN-GRK2. The results suggest a key role for GRKs in establishing the steady-state level of GPCR expression.DISCUSSIONCalcitonin is an important hormone in the regulation of serum calcium levels and bone mineral density through its effects on bone resorption and renal calcium excretion. Previous studies have not clearly defined the mechanisms involved in the regulation of CT receptor expression, in particular, agonist-mediated desensitization and down-regulation of these receptors (24Wada S. Martin T.J. Findlay D.M. Endocrinology. 1995; 136: 2611-2621Crossref PubMed Google Scholar, 25Wada S. Udagawa N. Nagata N. Martin T.J. Findlay D.M. Endocrinology. 1996; 137: 1042-1048Crossref PubMed Scopus (38) Google Scholar, 26Inoue D. Shih C. Galson R. Endocrinology. 1999; PubMed Google Scholar). In the current studies, we used CHO cells as a model to CT receptor expression by We found that retrovirally mediated gene transfer is a to expression of DN-GRK2. expression of DN-GRK2 phosphorylation by and CT-mediated desensitization of cAMP of cAMP in cells, however, to be have been from the loss in GRK2 activity and to to the increase in CT receptor of CT receptor signaling by DN-GRK2 expression was also in the phosphoinositide The of CT receptor number in cells was associated with an increase in mRNA for CT receptor These data suggest that the CT receptor, of the family of be by GRKs and that CT receptor mRNA and expression by GRK GRK isoforms that CT receptor signaling were not defined in our studies, but the data suggest that GRK2 may play an important role for desensitization of the Recent studies that various GPCRs be by multiple GRKs in Refs. 9Iacovelli L. Franchetti R. Grisolia D. De Blasi A. Mol. Pharmacol. 1999; 56: 316-324Crossref PubMed Scopus (53) Google Scholar, 10Lazari M.F. Liu X. Nakamura K. Benovic J.L. Ascoli M. Mol. Endocrinol. 1999; 13: 866-878Crossref PubMed Scopus (93) Google Scholar, and Premont R.T. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar). The receptor, of the be by expression of as as that of GRK2 or Premont R.T. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar). In the CHO cells that we in to GRK2, and also we found that the DN-GRK2 activity we an effect of the on activity of or as well. on to be we this is have several different to GRK expression Refs. 1Pitcher J.A. Freedman N.J. Lefkowitz R.J. Annu. Rev. Biochem. 1998; 67: 653-692Crossref PubMed Scopus (1060) Google Scholar, 2Krupnick J.G. Benovic J.L. Annu. Rev. Pharmacol. Toxicol. 1998; 38: 289-319Crossref PubMed Scopus (855) Google Scholar, 3Lefkowitz R.J. J. Biol. Chem. 1998; 273: 18677-18680Abstract Full Text Full Text PDF PubMed Scopus (903) Google Scholar, 4Hall R.A. Premont R.T. Lefkowitz R.J. J. Cell Biol. 1999; 145: 927-932Crossref PubMed Scopus (279) Google Scholar, 5Bunemann M. Hosey M.M. J. Physiol. ( Lond. ). 1999; 517: 5-23Crossref PubMed Scopus (163) Google Scholar). previous studies have involved of GRK The to GRK expression have use of a from GRK2 and studies in cells or from with a of a GRK We that use of a retrovirally expressed to other Thus, for is an of and of its in cells may be to but events (3Lefkowitz R.J. J. Biol. Chem. 1998; 273: 18677-18680Abstract Full Text Full Text PDF PubMed Scopus (903) Google Scholar, 4Hall R.A. Premont R.T. Lefkowitz R.J. J. Cell Biol. 1999; 145: 927-932Crossref PubMed Scopus (279) Google Scholar). Moreover, is not involved in the of GRK studies with not the role of which in and not for of stably cells. from is to and only from that have loss of expression of a of We that the retrovirally DN-GRK2 a to other of its to function of multiple GRK isoforms in GRK2 a in the of GRK its to stably expressing cells, such as we have used and the of the CHO cells used for the expression of the that we have may to GRK-mediated regulation of transfected findings in the CHO cells suggest that GRKs not only receptor desensitization but also play a role in the steady-state level of receptor of GRK to CT receptor desensitization phosphorylation of or of the in the of receptors. and be to the that by the such involved in GRK-mediated desensitization of the receptors, is not receptor phosphorylation by GRK receptor GRKs on the CT receptor through events to receptor phosphorylation and The evidence that DN-GRK2 cells have an increase in CT receptor mRNA that GRKs the of CT receptor In this is of that the of the current studies is on CT receptor, phosphoinositide or receptors, was also enhanced in the cells GRKs to expression of multiple of the current studies with CHO cells that these cells CT receptor and GRK2, and but not and We found that expression of DN-GRK2 by retrovirally mediated gene transfer CT receptor signaling cAMP and phosphoinositide in CHO cells, and desensitization of CT receptors. Moreover, CHO cells an in expression of CT receptors and receptor The findings provide a mechanistic for previous observations regarding agonist-mediated down-regulation of CT receptors, a that has been in resistance and escape from response to CT S. Udagawa N. Nagata N. Martin T.J. Findlay D.M. Endocrinology. 1996; 137: 1042-1048Crossref PubMed Scopus (38) Google Scholar, 26Inoue D. Shih C. Galson R. Endocrinology. 1999; PubMed Google Scholar). In addition, the data indicate that GRKs involved not only in desensitization of the CT receptor but also in the regulation of CT receptor We speculate that through effects on receptor GRKs to expression of CT receptor mRNA and in CHO cells and of GPCRs in other as well. Stimulation of G protein-coupled receptors (GPCRs)1 in the plasma membrane triggers two events, activation of G protein-mediated signal transduction pathways and, in parallel, a deactivation or desensitization of signaling. One component of this desensitization, in particular homologous, receptor-specific desensitization, is agonist-dependent phosphorylation of the receptor by specific G protein-coupled receptor kinases (GRKs). This phosphorylation event leads to the recruitment of cytosolic proteins, β-arrestins, to the receptor-signaling complex, the uncoupling of receptor from heterotrimeric G proteins, and loss of receptor responsiveness. Recent evidence indicates that GRKs and β-arrestins not only promote receptor uncoupling but also may directly participate in GPCR sequestration and the initiation of events leading to clathrin-coated pit-mediated internalization of receptors (for recent reviews see Refs. 1Pitcher J.A. Freedman N.J. Lefkowitz R.J. Annu. Rev. Biochem. 1998; 67: 653-692Crossref PubMed Scopus (1060) Google Scholar, 2Krupnick J.G. Benovic J.L. Annu. Rev. Pharmacol. Toxicol. 1998; 38: 289-319Crossref PubMed Scopus (855) Google Scholar, 3Lefkowitz R.J. J. Biol. Chem. 1998; 273: 18677-18680Abstract Full Text Full Text PDF PubMed Scopus (903) Google Scholar, 4Hall R.A. Premont R.T. Lefkowitz R.J. J. Cell Biol. 1999; 145: 927-932Crossref PubMed Scopus (279) Google Scholar, 5Bunemann M. Hosey M.M. J. Physiol. ( Lond. ). 1999; 517: 5-23Crossref PubMed Scopus (163) Google Scholar). In addition, GRK activation may be required to initiate certain events unrelated to receptor desensitization (3Lefkowitz R.J. J. Biol. Chem. 1998; 273: 18677-18680Abstract Full Text Full Text PDF PubMed Scopus (903) Google Scholar,4Hall R.A. Premont R.T. Lefkowitz R.J. J. Cell Biol. 1999; 145: 927-932Crossref PubMed Scopus (279) Google Scholar). least six different isoforms of the GRK family have been isolated. GRK2, formerly termed βARK1, is a widely expressed member of this family and has been shown to phosphorylate various GPCRs (1Pitcher J.A. Freedman N.J. Lefkowitz R.J. Annu. Rev. Biochem. 1998; 67: 653-692Crossref PubMed Scopus (1060) Google Scholar,2Krupnick J.G. Benovic J.L. Annu. Rev. Pharmacol. Toxicol. 1998; 38: 289-319Crossref PubMed Scopus (855) Google Scholar). GRK2 K220R, a dominant-negative GRK2 mutant (DN-GRK2) in which lysine at position 220 has been mutated to arginine to disrupt kinase activity (6Kong G. Penn R. Benovic J.L. J. Biol. Chem. 1994; 269: 13084-13087Abstract Full Text PDF PubMed Google Scholar), has been used to attenuate desensitization of several GPCR systems such as the β2-adrenergic receptor, α1B-adrenergic receptor, adenosine A2 receptor, thyrotropin receptor, follitropin receptor, and CCR2B receptor (6Kong G. Penn R. Benovic J.L. J. Biol. Chem. 1994; 269: 13084-13087Abstract Full Text PDF PubMed Google Scholar, 7Diviani D. Lattion A.L. Larbi N. Kunapuli P. Pronin A. Benovic J.L. Cotecchia S. J. Biol. Chem. 1996; 271: 5049-5058Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar, 8Mundell S.J. Benovic J.L. Kelly E. Mol. Pharmacol. 1997; 51: 991-998Crossref PubMed Scopus (63) Google Scholar, 9Iacovelli L. Franchetti R. Grisolia D. De Blasi A. Mol. Pharmacol. 1999; 56: 316-324Crossref PubMed Scopus (53) Google Scholar, 10Lazari M.F. Liu X. Nakamura K. Benovic J.L. Ascoli M. Mol. Endocrinol. 1999; 13: 866-878Crossref PubMed Scopus (93) Google Scholar, 11Aragay A.M. Mellado M. Frade J.M. Martin A.M. Jimenez-Sainz M.C Martinez- A, C Mayor F. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2985-2990Crossref PubMed Scopus (141) Google Scholar). date, however, relatively little is known about the role of GRKs in desensitization to peptide hormones, in particular, in the regulation and expression of endogenously expressed GPCRs. Thus, most studies of GRKs have involved the use of transfected cells expressing relatively nonphysiological levels of GPCRs. In the current studies, we utilized a model cell, Chinese hamster ovary (CHO) cells, which endogenously expresses calcitonin (CT) receptors, and we evaluated the role of endogenously expressed GRKs on signaling. We stably expressed DN-GRK2 in CHO cells using retrovirally mediated gene transfer and found that CT receptor expression and signaling were markedly enhanced by the DN-GRK2. The results suggest a key role for GRKs in establishing the steady-state level of GPCR is an important hormone in the regulation of serum calcium levels and bone mineral density through its effects on bone resorption and renal calcium excretion. Previous studies have not clearly defined the mechanisms involved in the regulation of CT receptor expression, in particular, agonist-mediated desensitization and down-regulation of these receptors (24Wada S. Martin T.J. Findlay D.M. Endocrinology. 1995; 136: 2611-2621Crossref PubMed Google Scholar, 25Wada S. Udagawa N. Nagata N. Martin T.J. Findlay D.M. Endocrinology. 1996; 137: 1042-1048Crossref PubMed Scopus (38) Google Scholar, 26Inoue D. Shih C. Galson R. Endocrinology. 1999; PubMed Google Scholar). In the current studies, we used CHO cells as a model to CT receptor expression by We found that retrovirally mediated gene transfer is a to expression of DN-GRK2. expression of DN-GRK2 phosphorylation by and CT-mediated desensitization of cAMP of cAMP in cells, however, to be have been from the loss in GRK2 activity and to to the increase in CT receptor of CT receptor signaling by DN-GRK2 expression was also in the phosphoinositide The of CT receptor number in cells was associated with an increase in mRNA for CT receptor These data suggest that the CT receptor, of the family of be by GRKs and that CT receptor mRNA and expression by GRK GRK isoforms that CT receptor signaling were not defined in our studies, but the data suggest that GRK2 may play an important role for desensitization of the Recent studies that various GPCRs be by multiple GRKs in Refs. 9Iacovelli L. Franchetti R. Grisolia D. De Blasi A. Mol. Pharmacol. 1999; 56: 316-324Crossref PubMed Scopus (53) Google Scholar, 10Lazari M.F. Liu X. Nakamura K. Benovic J.L. Ascoli M. Mol. Endocrinol. 1999; 13: 866-878Crossref PubMed Scopus (93) Google Scholar, and Premont R.T. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar). The receptor, of the be by expression of as as that of GRK2 or Premont R.T. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar). In the CHO cells that we in to GRK2, and also we found that the DN-GRK2 activity we an effect of the on activity of or as well. on to be we this is have several different to GRK expression Refs. 1Pitcher J.A. Freedman N.J. Lefkowitz R.J. Annu. Rev. Biochem. 1998; 67: 653-692Crossref PubMed Scopus (1060) Google Scholar, 2Krupnick J.G. Benovic J.L. Annu. Rev. Pharmacol. Toxicol. 1998; 38: 289-319Crossref PubMed Scopus (855) Google Scholar, 3Lefkowitz R.J. J. Biol. Chem. 1998; 273: 18677-18680Abstract Full Text Full Text PDF PubMed Scopus (903) Google Scholar, 4Hall R.A. Premont R.T. Lefkowitz R.J. J. Cell Biol. 1999; 145: 927-932Crossref PubMed Scopus (279) Google Scholar, 5Bunemann M. Hosey M.M. J. Physiol. ( Lond. ). 1999; 517: 5-23Crossref PubMed Scopus (163) Google Scholar). previous studies have involved of GRK The to GRK expression have use of a from GRK2 and studies in cells or from with a of a GRK We that use of a retrovirally expressed to other Thus, for is an of and of its in cells may be to but events (3Lefkowitz R.J. J. Biol. Chem. 1998; 273: 18677-18680Abstract Full Text Full Text PDF PubMed Scopus (903) Google Scholar, 4Hall R.A. Premont R.T. Lefkowitz R.J. J. Cell Biol. 1999; 145: 927-932Crossref PubMed Scopus (279) Google Scholar). Moreover, is not involved in the of GRK studies with not the role of which in and not for of stably cells. from is to and only from that have loss of expression of a of We that the retrovirally DN-GRK2 a to other of its to function of multiple GRK isoforms in GRK2 a in the of GRK its to stably expressing cells, such as we have used and the of the CHO cells used for the expression of the that we have may to GRK-mediated regulation of transfected findings in the CHO cells suggest that GRKs not only receptor desensitization but also play a role in the steady-state level of receptor of GRK to CT receptor desensitization phosphorylation of or of the in the of receptors. and be to the that by the such involved in GRK-mediated desensitization of the receptors, is not receptor phosphorylation by GRK receptor GRKs on the CT receptor through events to receptor phosphorylation and The evidence that DN-GRK2 cells have an increase in CT receptor mRNA that GRKs the of CT receptor In this is of that the of the current studies is on CT receptor, phosphoinositide or receptors, was also enhanced in the cells GRKs to expression of multiple of the current studies with CHO cells that these cells CT receptor and GRK2, and but not and We found that expression of DN-GRK2 by retrovirally mediated gene transfer CT receptor signaling cAMP and phosphoinositide in CHO cells, and desensitization of CT receptors. Moreover, CHO cells an in expression of CT receptors and receptor The findings provide a mechanistic for previous observations regarding agonist-mediated down-regulation of CT receptors, a that has been in resistance and escape from response to CT S. Udagawa N. Nagata N. Martin T.J. Findlay D.M. Endocrinology. 1996; 137: 1042-1048Crossref PubMed Scopus (38) Google Scholar, 26Inoue D. Shih C. Galson R. Endocrinology. 1999; PubMed Google Scholar). In addition, the data indicate that GRKs involved not only in desensitization of the CT receptor but also in the regulation of CT receptor We speculate that through effects on receptor GRKs to expression of CT receptor mRNA and in CHO cells and of GPCRs in other as well. is an important hormone in the regulation of serum calcium levels and bone mineral density through its effects on bone resorption and renal calcium excretion. Previous studies have not clearly defined the mechanisms involved in the regulation of CT receptor expression, in particular, agonist-mediated desensitization and down-regulation of these receptors (24Wada S. Martin T.J. Findlay D.M. Endocrinology. 1995; 136: 2611-2621Crossref PubMed Google Scholar, 25Wada S. Udagawa N. Nagata N. Martin T.J. Findlay D.M. Endocrinology. 1996; 137: 1042-1048Crossref PubMed Scopus (38) Google Scholar, 26Inoue D. Shih C. Galson R. Endocrinology. 1999; PubMed Google Scholar). In the current studies, we used CHO cells as a model to CT receptor expression by We found that retrovirally mediated gene transfer is a to expression of DN-GRK2. expression of DN-GRK2 phosphorylation by and CT-mediated desensitization of cAMP of cAMP in cells, however, to be have been from the loss in GRK2 activity and to to the increase in CT receptor of CT receptor signaling by DN-GRK2 expression was also in the phosphoinositide The of CT receptor number in cells was associated with an increase in mRNA for CT receptor These data suggest that the CT receptor, of the family of be by GRKs and that CT receptor mRNA and expression by GRK The GRK isoforms that CT receptor signaling were not defined in our studies, but the data suggest that GRK2 may play an important role for desensitization of the Recent studies that various GPCRs be by multiple GRKs in Refs. 9Iacovelli L. Franchetti R. Grisolia D. De Blasi A. Mol. Pharmacol. 1999; 56: 316-324Crossref PubMed Scopus (53) Google Scholar, 10Lazari M.F. Liu X. Nakamura K. Benovic J.L. Ascoli M. Mol. Endocrinol. 1999; 13: 866-878Crossref PubMed Scopus (93) Google Scholar, and Premont R.T. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar). The receptor, of the be by expression of as as that of GRK2 or Premont R.T. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar). In the CHO cells that we in to GRK2, and also we found that the DN-GRK2 activity we an effect of the on activity of or as well. on to be we this is Previous have several different to GRK expression Refs. 1Pitcher J.A. Freedman N.J. Lefkowitz R.J. Annu. Rev. Biochem. 1998; 67: 653-692Crossref PubMed Scopus (1060) Google Scholar, 2Krupnick J.G. Benovic J.L. Annu. Rev. Pharmacol. Toxicol. 1998; 38: 289-319Crossref PubMed Scopus (855) Google Scholar, 3Lefkowitz R.J. J. Biol. Chem. 1998; 273: 18677-18680Abstract Full Text Full Text PDF PubMed Scopus (903) Google Scholar, 4Hall R.A. Premont R.T. Lefkowitz R.J. J. Cell Biol. 1999; 145: 927-932Crossref PubMed Scopus (279) Google Scholar, 5Bunemann M. Hosey M.M. J. Physiol. ( Lond. ). 1999; 517: 5-23Crossref PubMed Scopus (163) Google Scholar). previous studies have involved of GRK The to GRK expression have use of a from GRK2 and studies in cells or from with a of a GRK We that use of a retrovirally expressed to other Thus, for is an of and of its in cells may be to but events (3Lefkowitz R.J. J. Biol. Chem. 1998; 273: 18677-18680Abstract Full Text Full Text PDF PubMed Scopus (903) Google Scholar, 4Hall R.A. Premont R.T. Lefkowitz R.J. J. Cell Biol. 1999; 145: 927-932Crossref PubMed Scopus (279) Google Scholar). Moreover, is not involved in the of GRK studies with not the role of which in and not for of stably cells. from is to and only from that have loss of expression of a of We that the retrovirally DN-GRK2 a to other of its to function of multiple GRK isoforms in GRK2 a in the of GRK its to stably expressing cells, such as we have used and the of the CHO cells used for the expression of the that we have may to GRK-mediated regulation of transfected GPCRs. findings in the CHO cells suggest that GRKs not only receptor desensitization but also play a role in the steady-state level of receptor of GRK to CT receptor desensitization phosphorylation of or of the in the of receptors. and be to the that by the such involved in GRK-mediated desensitization of the receptors, is not receptor phosphorylation by GRK receptor GRKs on the CT receptor through events to receptor phosphorylation and The evidence that DN-GRK2 cells have an increase in CT receptor mRNA that GRKs the of CT receptor In this is of that the of the current studies is on CT receptor, phosphoinositide or receptors, was also enhanced in the cells GRKs to expression of multiple of GPCRs. In the current studies with CHO cells that these cells CT receptor and GRK2, and but not and We found that expression of DN-GRK2 by retrovirally mediated gene transfer CT receptor signaling cAMP and phosphoinositide in CHO cells, and desensitization of CT receptors. Moreover, CHO cells an in expression of CT receptors and receptor The findings provide a mechanistic for previous observations regarding agonist-mediated down-regulation of CT receptors, a that has been in resistance and escape from response to CT S. Udagawa N. Nagata N. Martin T.J. Findlay D.M. Endocrinology. 1996; 137: 1042-1048Crossref PubMed Scopus (38) Google Scholar, 26Inoue D. Shih C. Galson R. Endocrinology. 1999; PubMed Google Scholar). In addition, the data indicate that GRKs involved not only in desensitization of the CT receptor but also in the regulation of CT receptor We speculate that through effects on receptor GRKs to expression of CT receptor mRNA and in CHO cells and of GPCRs in other as well. We to Benovic for the GRK2 Lefkowitz for for and J. for GRK2 for CHO cells, and for and for of and and for regarding phosphorylation and We also for for CHO and for in of this