FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2034172498> ?p ?o ?g. }

• W2034172498 endingPage "29440" @default.
• W2034172498 startingPage "29433" @default.
• W2034172498 abstract "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 D.L. Goldring S.R. Horne W.C. Baron R. Endocrinology. 1999; 140: 1060-1068Crossref PubMed Google Scholar). In the current studies, we used CHO 10001 cells as a model to examine CT receptor expression by GRKs. We found that retrovirally mediated gene transfer is a useful means to establish stable expression of DN-GRK2. Stable expression of DN-GRK2 inhibited GRK2-mediated substrate phosphorylation by approximately 40% and impaired CT-mediated desensitization of cAMP formation. Potentiation of cAMP generation in DN-GRK2-expressing cells, however, appeared to be much greater than what would have been expected from the loss in GRK2 phosphorylating activity and appeared to relate to the increase in CT receptor number. Enhancement of CT receptor signaling by DN-GRK2 expression was also observed in the phosphoinositide pathway. The up-regulation of CT receptor number in DN-GRK2-expressing cells was associated with an increase in mRNA for CT receptor C1a. These data suggest that the CT receptor, one of the Class II family of GPCRs, can be regulated by GRKs and that CT receptor mRNA and protein expression are negatively influenced by GRK activity.The GRK isoforms that regulate CT receptor signaling were not precisely defined in our studies, but the data suggest that GRK2 may play an important role for desensitization of the receptor. Recent studies reveal that various GPCRs can be regulated by multiple GRKs in vitro (e.g. 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 27Shetzline M.A. Premont R.T. Walker J.K. Vigna S.R. Caron M.G. J. Biol. Chem. 1998; 273: 6756-6762Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). The secretin receptor, another of the class II GPCRs, can be desensitized by expression of GRK5 as well as that of GRK2 or GRK3 (27Shetzline M.A. Premont R.T. Walker J.K. Vigna S.R. Caron M.G. J. Biol. Chem. 1998; 273: 6756-6762Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). In the CHO cells that we used, in addition to GRK2, GRK6 and GRK5 are also expressed. Although we found that the K220R DN-GRK2 construct inhibited GRK2-mediated activity (Fig. 3), we cannot rule out an effect of the DN construct on activity of GRK5 or GRK6 as well. Indeed, based on structural similarities, to be described below, we believe this is quite likely.Previous workers have employed several different techniques to modulate GRK expression (e.g. 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). Many previous studies have involved overexpression of GRK isoforms. The more limited efforts designed to inhibit GRK expression have included use of a carboxyl-terminal fragment from GRK2 (termed βARKct), antisense oligonucleotides, and studies in cells or tissues from mice with a knockout of a GRK isoform. We believe that use of a retrovirally expressed DN construct offers advantages relative to those other methods. Thus, for example, βARKct is an inhibitor of Gβγ and some of its actions in cells may be attributable to Gβγ-dependent, but GRK-independent, 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, because Gβγ is not involved in the action of all GRK isoforms, studies with βRKct will not assess the role of Gβγ-independent isoforms. Antisense oligonucleotides, which are generally isoform-selective, are primarily useful in acute experiments and not for generation of stably inhibited cells. Material from knockout animals is limited thus far to murine tissues and generally only from heterozygotic animals that have loss of expression of a single type of GRK. We believe that the retrovirally engineered DN-GRK2 construct provides a useful complementary approach to those other methods because of its theoretical ability to block function of multiple GRK isoforms (the K220R in GRK2 represents a conserved region in the catalytic domain ATP-binding site of all GRK isoforms), its potential utility to generate stably expressing cells, such as those we have used here, and the rather widespread tropism of the retroviral vector. Since CHO cells are often used for the heterologous expression of GPCRs, the stable cell line that we have developed may prove useful to examine GRK-mediated regulation of transfected GPCRs.Our findings in the DN-GRK CHO cells strongly suggest that GRKs not only regulate receptor desensitization but also play a role in the steady-state level of receptor expression. Activity of GRK to regulate CT receptor desensitization presumably reflects phosphorylation of one or more of the 8 serine/threonine residues in the carboxyl-terminal portion of C1a receptors. Mutagenesis and related approaches will be necessary to define the precise sites that are regulated by the GRKs. Although such sites likely are involved in GRK-mediated desensitization of the receptors, it is not clear how receptor phosphorylation by GRK would regulate receptor expression. Presumably, GRKs impact on the CT receptor life cycle through events subsequent to receptor phosphorylation and internalization. The evidence that DN-GRK2 cells have an increase in CT receptor mRNA suggests that GRKs regulate the transcription and/or turnover of CT receptor mRNA. In this regard, it is of further interest that although the focus of the current studies is on CT receptor, ATP-mediated phosphoinositide hydrolysis, presumably via one or more P2Y receptors, was also enhanced in the DN cells (Fig. 8). Perhaps GRKs are able to influence expression of multiple types of GPCRs.In summary, the current studies with CHO cells show that these cells express CT receptor C1a and GRK2, GRK5 and GRK6, but not GRK3 and GRK4. We found that stable expression of DN-GRK2 by retrovirally mediated gene transfer inhibited GRK2-promoted substrate phosphorylation, potentiated CT receptor signaling (both cAMP generation and phosphoinositide hydrolysis) in CHO cells, and blunted desensitization of CT receptors. Moreover, DN-GRK2-expressing CHO cells had an up-regulation in expression of CT receptors and receptor mRNA. The findings thus provide a mechanistic explanation for previous observations regarding agonist-mediated down-regulation of CT receptors, a phenomenon that has been implicated in resistance and escape from response to CT (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 D.L. Goldring S.R. Horne W.C. Baron R. Endocrinology. 1999; 140: 1060-1068Crossref PubMed Google Scholar). In addition, the data indicate that GRKs are involved not only in desensitization of the CT receptor but also in the regulation of CT receptor expression. We speculate that through their effects on receptor phosphorylation, GRKs are able to inhibit expression of CT receptor mRNA and protein in CHO cells and perhaps more generally of GPCRs in other cell types 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). 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 D.L. Goldring S.R. Horne W.C. Baron R. Endocrinology. 1999; 140: 1060-1068Crossref PubMed Google Scholar). In the current studies, we used CHO 10001 cells as a model to examine CT receptor expression by GRKs. We found that retrovirally mediated gene transfer is a useful means to establish stable expression of DN-GRK2. Stable expression of DN-GRK2 inhibited GRK2-mediated substrate phosphorylation by approximately 40% and impaired CT-mediated desensitization of cAMP formation. Potentiation of cAMP generation in DN-GRK2-expressing cells, however, appeared to be much greater than what would have been expected from the loss in GRK2 phosphorylating activity and appeared to relate to the increase in CT receptor number. Enhancement of CT receptor signaling by DN-GRK2 expression was also observed in the phosphoinositide pathway. The up-regulation of CT receptor number in DN-GRK2-expressing cells was associated with an increase in mRNA for CT receptor C1a. These data suggest that the CT receptor, one of the Class II family of GPCRs, can be regulated by GRKs and that CT receptor mRNA and protein expression are negatively influenced by GRK activity.The GRK isoforms that regulate CT receptor signaling were not precisely defined in our studies, but the data suggest that GRK2 may play an important role for desensitization of the receptor. Recent studies reveal that various GPCRs can be regulated by multiple GRKs in vitro (e.g. 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 27Shetzline M.A. Premont R.T. Walker J.K. Vigna S.R. Caron M.G. J. Biol. Chem. 1998; 273: 6756-6762Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). The secretin receptor, another of the class II GPCRs, can be desensitized by expression of GRK5 as well as that of GRK2 or GRK3 (27Shetzline M.A. Premont R.T. Walker J.K. Vigna S.R. Caron M.G. J. Biol. Chem. 1998; 273: 6756-6762Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). In the CHO cells that we used, in addition to GRK2, GRK6 and GRK5 are also expressed. Although we found that the K220R DN-GRK2 construct inhibited GRK2-mediated activity (Fig. 3), we cannot rule out an effect of the DN construct on activity of GRK5 or GRK6 as well. Indeed, based on structural similarities, to be described below, we believe this is quite likely.Previous workers have employed several different techniques to modulate GRK expression (e.g. 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). Many previous studies have involved overexpression of GRK isoforms. The more limited efforts designed to inhibit GRK expression have included use of a carboxyl-terminal fragment from GRK2 (termed βARKct), antisense oligonucleotides, and studies in cells or tissues from mice with a knockout of a GRK isoform. We believe that use of a retrovirally expressed DN construct offers advantages relative to those other methods. Thus, for example, βARKct is an inhibitor of Gβγ and some of its actions in cells may be attributable to Gβγ-dependent, but GRK-independent, 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, because Gβγ is not involved in the action of all GRK isoforms, studies with βRKct will not assess the role of Gβγ-independent isoforms. Antisense oligonucleotides, which are generally isoform-selective, are primarily useful in acute experiments and not for generation of stably inhibited cells. Material from knockout animals is limited thus far to murine tissues and generally only from heterozygotic animals that have loss of expression of a single type of GRK. We believe that the retrovirally engineered DN-GRK2 construct provides a useful complementary approach to those other methods because of its theoretical ability to block function of multiple GRK isoforms (the K220R in GRK2 represents a conserved region in the catalytic domain ATP-binding site of all GRK isoforms), its potential utility to generate stably expressing cells, such as those we have used here, and the rather widespread tropism of the retroviral vector. Since CHO cells are often used for the heterologous expression of GPCRs, the stable cell line that we have developed may prove useful to examine GRK-mediated regulation of transfected GPCRs.Our findings in the DN-GRK CHO cells strongly suggest that GRKs not only regulate receptor desensitization but also play a role in the steady-state level of receptor expression. Activity of GRK to regulate CT receptor desensitization presumably reflects phosphorylation of one or more of the 8 serine/threonine residues in the carboxyl-terminal portion of C1a receptors. Mutagenesis and related approaches will be necessary to define the precise sites that are regulated by the GRKs. Although such sites likely are involved in GRK-mediated desensitization of the receptors, it is not clear how receptor phosphorylation by GRK would regulate receptor expression. Presumably, GRKs impact on the CT receptor life cycle through events subsequent to receptor phosphorylation and internalization. The evidence that DN-GRK2 cells have an increase in CT receptor mRNA suggests that GRKs regulate the transcription and/or turnover of CT receptor mRNA. In this regard, it is of further interest that although the focus of the current studies is on CT receptor, ATP-mediated phosphoinositide hydrolysis, presumably via one or more P2Y receptors, was also enhanced in the DN cells (Fig. 8). Perhaps GRKs are able to influence expression of multiple types of GPCRs.In summary, the current studies with CHO cells show that these cells express CT receptor C1a and GRK2, GRK5 and GRK6, but not GRK3 and GRK4. We found that stable expression of DN-GRK2 by retrovirally mediated gene transfer inhibited GRK2-promoted substrate phosphorylation, potentiated CT receptor signaling (both cAMP generation and phosphoinositide hydrolysis) in CHO cells, and blunted desensitization of CT receptors. Moreover, DN-GRK2-expressing CHO cells had an up-regulation in expression of CT receptors and receptor mRNA. The findings thus provide a mechanistic explanation for previous observations regarding agonist-mediated down-regulation of CT receptors, a phenomenon that has been implicated in resistance and escape from response to CT (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 D.L. Goldring S.R. Horne W.C. Baron R. Endocrinology. 1999; 140: 1060-1068Crossref PubMed Google Scholar). In addition, the data indicate that GRKs are involved not only in desensitization of the CT receptor but also in the regulation of CT receptor expression. We speculate that through their effects on receptor phosphorylation, GRKs are able to inhibit expression of CT receptor mRNA and protein in CHO cells and perhaps more generally of GPCRs in other cell types as well. Calcitonin 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 D.L. Goldring S.R. Horne W.C. Baron R. Endocrinology. 1999; 140: 1060-1068Crossref PubMed Google Scholar). In the current studies, we used CHO 10001 cells as a model to examine CT receptor expression by GRKs. We found that retrovirally mediated gene transfer is a useful means to establish stable expression of DN-GRK2. Stable expression of DN-GRK2 inhibited GRK2-mediated substrate phosphorylation by approximately 40% and impaired CT-mediated desensitization of cAMP formation. Potentiation of cAMP generation in DN-GRK2-expressing cells, however, appeared to be much greater than what would have been expected from the loss in GRK2 phosphorylating activity and appeared to relate to the increase in CT receptor number. Enhancement of CT receptor signaling by DN-GRK2 expression was also observed in the phosphoinositide pathway. The up-regulation of CT receptor number in DN-GRK2-expressing cells was associated with an increase in mRNA for CT receptor C1a. These data suggest that the CT receptor, one of the Class II family of GPCRs, can be regulated by GRKs and that CT receptor mRNA and protein expression are negatively influenced by GRK activity. The GRK isoforms that regulate CT receptor signaling were not precisely defined in our studies, but the data suggest that GRK2 may play an important role for desensitization of the receptor. Recent studies reveal that various GPCRs can be regulated by multiple GRKs in vitro (e.g. 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 27Shetzline M.A. Premont R.T. Walker J.K. Vigna S.R. Caron M.G. J. Biol. Chem. 1998; 273: 6756-6762Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). The secretin receptor, another of the class II GPCRs, can be desensitized by expression of GRK5 as well as that of GRK2 or GRK3 (27Shetzline M.A. Premont R.T. Walker J.K. Vigna S.R. Caron M.G. J. Biol. Chem. 1998; 273: 6756-6762Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). In the CHO cells that we used, in addition to GRK2, GRK6 and GRK5 are also expressed. Although we found that the K220R DN-GRK2 construct inhibited GRK2-mediated activity (Fig. 3), we cannot rule out an effect of the DN construct on activity of GRK5 or GRK6 as well. Indeed, based on structural similarities, to be described below, we believe this is quite likely. Previous workers have employed several different techniques to modulate GRK expression (e.g. 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). Many previous studies have involved overexpression of GRK isoforms. The more limited efforts designed to inhibit GRK expression have included use of a carboxyl-terminal fragment from GRK2 (termed βARKct), antisense oligonucleotides, and studies in cells or tissues from mice with a knockout of a GRK isoform. We believe that use of a retrovirally expressed DN construct offers advantages relative to those other methods. Thus, for example, βARKct is an inhibitor of Gβγ and some of its actions in cells may be attributable to Gβγ-dependent, but GRK-independent, 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, because Gβγ is not involved in the action of all GRK isoforms, studies with βRKct will not assess the role of Gβγ-independent isoforms. Antisense oligonucleotides, which are generally isoform-selective, are primarily useful in acute experiments and not for generation of stably inhibited cells. Material from knockout animals is limited thus far to murine tissues and generally only from heterozygotic animals that have loss of expression of a single type of GRK. We believe that the retrovirally engineered DN-GRK2 construct provides a useful complementary approach to those other methods because of its theoretical ability to block function of multiple GRK isoforms (the K220R in GRK2 represents a conserved region in the catalytic domain ATP-binding site of all GRK isoforms), its potential utility to generate stably expressing cells, such as those we have used here, and the rather widespread tropism of the retroviral vector. Since CHO cells are often used for the heterologous expression of GPCRs, the stable cell line that we have developed may prove useful to examine GRK-mediated regulation of transfected GPCRs. Our findings in the DN-GRK CHO cells strongly suggest that GRKs not only regulate receptor desensitization but also play a role in the steady-state level of receptor expression. Activity of GRK to regulate CT receptor desensitization presumably reflects phosphorylation of one or more of the 8 serine/threonine residues in the carboxyl-terminal portion of C1a receptors. Mutagenesis and related approaches will be necessary to define the precise sites that are regulated by the GRKs. Although such sites likely are involved in GRK-mediated desensitization of the receptors, it is not clear how receptor phosphorylation by GRK would regulate receptor expression. Presumably, GRKs impact on the CT receptor life cycle through events subsequent to receptor phosphorylation and internalization. The evidence that DN-GRK2 cells have an increase in CT receptor mRNA suggests that GRKs regulate the transcription and/or turnover of CT receptor mRNA. In this regard, it is of further interest that although the focus of the current studies is on CT receptor, ATP-mediated phosphoinositide hydrolysis, presumably via one or more P2Y receptors, was also enhanced in the DN cells (Fig. 8). Perhaps GRKs are able to influence expression of multiple types of GPCRs. In summary, the current studies with CHO cells show that these cells express CT receptor C1a and GRK2, GRK5 and GRK6, but not GRK3 and GRK4. We found that stable expression of DN-GRK2 by retrovirally mediated gene transfer inhibited GRK2-promoted substrate phosphorylation, potentiated CT receptor signaling (both cAMP generation and phosphoinositide hydrolysis) in CHO cells, and blunted desensitization of CT receptors. Moreover, DN-GRK2-expressing CHO cells had an up-regulation in expression of CT receptors and receptor mRNA. The findings thus provide a mechanistic explanation for previous observations regarding agonist-mediated down-regulation of CT receptors, a phenomenon that has been implicated in resistance and escape from response to CT (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 D.L. Goldring S.R. Horne W.C. Baron R. Endocrinology. 1999; 140: 1060-1068Crossref PubMed Google Scholar). In addition, the data indicate that GRKs are involved not only in desensitization of the CT receptor but also in the regulation of CT receptor expression. We speculate that through their effects on receptor phosphorylation, GRKs are able to inhibit expression of CT receptor mRNA and protein in CHO cells and perhaps more generally of GPCRs in other cell types as well. We are grateful to Dr. Jeffrey Benovic for the K220R GRK2 construct; Dr. Robert Lefkowitz for rat GRK3 cDNA; Dr. Jean-Marc Elalouf for rat GRK4 and rat GRK6 cDNAs; Dr. Richard J. Hughes for murine GRK2 cDNA; Dr. Michael Gottesman for CHO 10001 cells, Ryan Adams and Dr. Alexandra Newton for rhodopsin kinase; Drs. Sam Farlow and Lawrence Goldstein for preparation of tubulin; and Drs. David Williams and Leonard Deftos for helpful discussions regarding rhodopsin phosphorylation and calcitonin, respectively. We also thank Brain Torres for expert technical assistance, Jenny Truong for CHO cell maintenance, and Laurie Cartlidge for assistance in preparation of this manuscript." @default.
• W2034172498 created "2016-06-24" @default.
• W2034172498 creator A5019470020 @default.
• W2034172498 creator A5055677266 @default.
• W2034172498 date "2000-09-01" @default.
• W2034172498 modified "2023-10-16" @default.
• W2034172498 title "Retrovirally Mediated Transfer of a G Protein-coupled Receptor Kinase (GRK) Dominant-negative Mutant Enhances Endogenous Calcitonin Receptor Signaling in Chinese Hamster Ovary Cells" @default.
• W2034172498 cites W1593834989 @default.
• W2034172498 cites W1929814565 @default.
• W2034172498 cites W1963640930 @default.
• W2034172498 cites W1969497210 @default.
• W2034172498 cites W1980445909 @default.
• W2034172498 cites W1980674121 @default.
• W2034172498 cites W1998163989 @default.
• W2034172498 cites W2001289439 @default.
• W2034172498 cites W2025663580 @default.
• W2034172498 cites W2055863819 @default.
• W2034172498 cites W2062870899 @default.
• W2034172498 cites W2066410164 @default.
• W2034172498 cites W2089670042 @default.
• W2034172498 cites W2090020265 @default.
• W2034172498 cites W2110353582 @default.
• W2034172498 cites W2113122754 @default.
• W2034172498 cites W2125468282 @default.
• W2034172498 cites W2163741449 @default.
• W2034172498 cites W2188184596 @default.
• W2034172498 cites W2471042929 @default.
• W2034172498 cites W2614496215 @default.
• W2034172498 cites W942133498 @default.
• W2034172498 doi "https://doi.org/10.1074/jbc.m003413200" @default.
• W2034172498 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/10889199" @default.
• W2034172498 hasPublicationYear "2000" @default.
• W2034172498 type Work @default.
• W2034172498 sameAs 2034172498 @default.
• W2034172498 citedByCount "25" @default.
• W2034172498 countsByYear W20341724982013 @default.
• W2034172498 countsByYear W20341724982015 @default.
• W2034172498 countsByYear W20341724982019 @default.
• W2034172498 crossrefType "journal-article" @default.
• W2034172498 hasAuthorship W2034172498A5019470020 @default.
• W2034172498 hasAuthorship W2034172498A5055677266 @default.
• W2034172498 hasBestOaLocation W20341724981 @default.
• W2034172498 hasConcept C104317684 @default.
• W2034172498 hasConcept C118303440 @default.
• W2034172498 hasConcept C134018914 @default.
• W2034172498 hasConcept C135285700 @default.
• W2034172498 hasConcept C143065580 @default.
• W2034172498 hasConcept C153911025 @default.
• W2034172498 hasConcept C163170386 @default.
• W2034172498 hasConcept C16613235 @default.
• W2034172498 hasConcept C170493617 @default.
• W2034172498 hasConcept C175656101 @default.
• W2034172498 hasConcept C184235292 @default.
• W2034172498 hasConcept C185592680 @default.
• W2034172498 hasConcept C2778575167 @default.
• W2034172498 hasConcept C2779627488 @default.
• W2034172498 hasConcept C3019218413 @default.
• W2034172498 hasConcept C55493867 @default.
• W2034172498 hasConcept C62478195 @default.
• W2034172498 hasConcept C86803240 @default.
• W2034172498 hasConcept C92316933 @default.
• W2034172498 hasConcept C95444343 @default.
• W2034172498 hasConcept C97029542 @default.
• W2034172498 hasConceptScore W2034172498C104317684 @default.
• W2034172498 hasConceptScore W2034172498C118303440 @default.
• W2034172498 hasConceptScore W2034172498C134018914 @default.
• W2034172498 hasConceptScore W2034172498C135285700 @default.
• W2034172498 hasConceptScore W2034172498C143065580 @default.
• W2034172498 hasConceptScore W2034172498C153911025 @default.
• W2034172498 hasConceptScore W2034172498C163170386 @default.
• W2034172498 hasConceptScore W2034172498C16613235 @default.
• W2034172498 hasConceptScore W2034172498C170493617 @default.
• W2034172498 hasConceptScore W2034172498C175656101 @default.
• W2034172498 hasConceptScore W2034172498C184235292 @default.
• W2034172498 hasConceptScore W2034172498C185592680 @default.
• W2034172498 hasConceptScore W2034172498C2778575167 @default.
• W2034172498 hasConceptScore W2034172498C2779627488 @default.
• W2034172498 hasConceptScore W2034172498C3019218413 @default.
• W2034172498 hasConceptScore W2034172498C55493867 @default.
• W2034172498 hasConceptScore W2034172498C62478195 @default.
• W2034172498 hasConceptScore W2034172498C86803240 @default.
• W2034172498 hasConceptScore W2034172498C92316933 @default.
• W2034172498 hasConceptScore W2034172498C95444343 @default.
• W2034172498 hasConceptScore W2034172498C97029542 @default.
• W2034172498 hasIssue "38" @default.
• W2034172498 hasLocation W20341724981 @default.
• W2034172498 hasOpenAccess W2034172498 @default.
• W2034172498 hasPrimaryLocation W20341724981 @default.
• W2034172498 hasRelatedWork W1983368465 @default.
• W2034172498 hasRelatedWork W1986085088 @default.
• W2034172498 hasRelatedWork W1986329711 @default.
• W2034172498 hasRelatedWork W2015050496 @default.
• W2034172498 hasRelatedWork W2037857433 @default.
• W2034172498 hasRelatedWork W2394909441 @default.
• W2034172498 hasRelatedWork W2752752156 @default.
• W2034172498 hasRelatedWork W3029054931 @default.
• W2034172498 hasRelatedWork W3138702158 @default.
• W2034172498 hasRelatedWork W4200387587 @default.

• next