Visualization of Agonist-induced Sequestration and Down-regulation of a Green Fluorescent Protein-tagged β2-Adrenergic Receptor

Abstract

To date, the visualization of β2-adrenergic receptor (β2AR) trafficking has been largely limited to immunocytochemical analyses of acute internalization events of epitope-tagged receptors in various transfection systems. The development of a β2AR conjugated with green fluorescent protein (β2AR-GFP) provides the opportunity for a more extensive optical analysis of β2AR sequestration, down-regulation, and recycling in cells. Here we demonstrate that stable expression of β2AR-GFP in HeLa cells enables a detailed temporal and spatial analysis of these events. Time-dependent colocalization of β2AR-GFP with rhodamine-labeled transferrin and rhodamine-labeled dextran following agonist exposure demonstrates receptor distribution to early endosomes (sequestration) and lysosomes (down-regulation), respectively. The observed temporal distribution of β2AR-GFP was consistent with measures of receptor sequestration and down-regulation generated by radioligand-receptor binding assays. Cells stimulated with different β-agonists revealed time courses of β2AR-GFP redistribution reflective of the intrinsic activity of each agonist. To date, the visualization of β2-adrenergic receptor (β2AR) trafficking has been largely limited to immunocytochemical analyses of acute internalization events of epitope-tagged receptors in various transfection systems. The development of a β2AR conjugated with green fluorescent protein (β2AR-GFP) provides the opportunity for a more extensive optical analysis of β2AR sequestration, down-regulation, and recycling in cells. Here we demonstrate that stable expression of β2AR-GFP in HeLa cells enables a detailed temporal and spatial analysis of these events. Time-dependent colocalization of β2AR-GFP with rhodamine-labeled transferrin and rhodamine-labeled dextran following agonist exposure demonstrates receptor distribution to early endosomes (sequestration) and lysosomes (down-regulation), respectively. The observed temporal distribution of β2AR-GFP was consistent with measures of receptor sequestration and down-regulation generated by radioligand-receptor binding assays. Cells stimulated with different β-agonists revealed time courses of β2AR-GFP redistribution reflective of the intrinsic activity of each agonist. Upon agonist stimulation, β2-adrenergic receptors (β2ARs) 1The abbreviations used are: β2AR, β2-adrenergic receptor; GFP, green fluorescent protein; PCR, polymerase chain reaction; PBS, phosphate-buffered saline. 1The abbreviations used are: β2AR, β2-adrenergic receptor; GFP, green fluorescent protein; PCR, polymerase chain reaction; PBS, phosphate-buffered saline. are rapidly desensitized by receptor phosphorylation (1Sterne-Marr R. Benovic J.L. Litwack G. Vitamins and Hormones. Academic Press, New York1995: 193-234Google Scholar). Receptor phosphorylation by G protein-coupled receptor kinases and subsequent binding of non-visual arrestins initiates the internalization of β2ARs via clathrin-coated pits (2Ferguson S.S.G. Downey III, W.E. Colapietro A.M. Barak L.S. Menard L. Caron M.G. Science. 1996; 271: 363-365Crossref PubMed Scopus (839) Google Scholar, 3Goodman O.B.J. Krupnick J.G. Santini F. Gurevich V.V. Penn R.B. Gagnon A.W. Keen J.H. Benovic J.L. Nature. 1996; 383: 447-450Crossref PubMed Scopus (1154) Google Scholar). Previous studies have demonstrated that internalized receptors have multiple potential fates. One such fate is the recycling of internalized receptors to the plasma membrane, presumably completing an ill defined “resensitization ” process involving β2AR dephosphorylation in an endosomal compartment (4Pippig S. Andexinger S. Lohse M. Mol. Pharmacol. 1995; 47: 666-676PubMed Google Scholar, 5Krueger K.M. Daaka Y. Pitcher J.A. Lefkowitz R.J. J. Biol. Chem. 1997; 272: 5-8Abstract Full Text Full Text PDF PubMed Scopus (312) Google Scholar). Depending upon the duration of agonist exposure, internalized β2ARs may ultimately appear “lost” or destroyed (undetectable by radioligand binding), ostensibly trafficking to lysosomes where they are degraded. Previously, the analysis of these events has been heavily dependent on biochemical and pharmacological approaches.Agonist-mediated subcellular redistribution of β2ARs was initially inferred from ligand binding studies (6Toews M.L. Liang M. Perkins J.P. Mol. Pharmacol. 1987; 32: 737-742PubMed Google Scholar, 7Kurz J.B. Perkins J.P. Mol. Pharmacol. 1992; 41: 375-381PubMed Google Scholar) and the coincident migration of β2ARs with enzyme markers (8Chuang D.M. Biochem. Biophys. Res. Commun. 1982; 105: 1466-1472Crossref PubMed Scopus (13) Google Scholar, 9Waldo G.L. Northup J.K. Perkins J.P. Harden T.K. J. Biol. Chem. 1983; 258: 13900-13908Abstract Full Text PDF PubMed Google Scholar) or epidermal growth factor (10Wakshull E. Hertel C. O'Keefe E.J. Perkins J.P. J. Cell. Biochem. 1985; 29: 127-141Crossref PubMed Scopus (10) Google Scholar, 11Liao J.-F. Perkins J.P. Mol. Pharmacol. 1993; 44: 364-370PubMed Google Scholar) into subcellular fractions resolved through centrifugation. However, direct visualization of β2AR trafficking events remained lacking until von Zastrow and Kobilka (12von Zastrow M. Kobilka B.K. J. Biol. Chem. 1992; 267: 3530-3538Abstract Full Text PDF PubMed Google Scholar) provided immunocytochemical evidence of rapid, agonist-induced redistribution of epitope-tagged β2ARs into small, punctate accumulations within the cytoplasm. The time course of β2AR redistribution assessed by confocal microscopy paralleled that of β2AR sequestration measured by radioligand binding. Importantly, internalized β2ARs colocalized with transferrin receptors, suggesting that sequestered β2ARs undergo processing through endosomal compartments in a manner similar to that observed for constitutively internalized receptors.Advances in the development of proteins conjugated with green fluorescent protein (GFP) have since provided the opportunity for real time optical analysis of protein trafficking events in individual cells (13Prasher D.C. Eckenrode V.K. Ward W.W. Prendergast F.G. Cormier M.J. Gene (Amst.). 1992; 111: 229-233Crossref PubMed Scopus (1748) Google Scholar, 14Kain S.R. Adams M. Kondepudi A. Yang T.-T. Ward W.W. Kitts P. BioTechniques. 1995; 19: 650-654PubMed Google Scholar, 15Inouye S. Tsuji F.I. FEBS Lett. 1994; 341: 277-280Crossref PubMed Scopus (353) Google Scholar). Green fluorescent protein is a naturally occurring protein isolated from several different species of jellyfish (Aequoria) and sea cucumbers (Renilla). When expressed in cells, proteins conjugated with GFP may be visualized with routine fluorescent microscopy without the need to fix cells. Recently, Barak et al. (16Barak L.S. Ferguson S.S.G. Zhang J. Martenson C. Meyer T. Caron M.G. Mol. Pharmacol. 1997; 51: 177-184Crossref PubMed Scopus (201) Google Scholar) established the utility of a β2AR-GFP fusion protein in visualizing agonist-mediated β2AR internalization in HEK293 cells transiently expressing the construct. This study also demonstrated that β2AR-GFP is fully functional with regard to ligand binding and adenylyl cyclase stimulation and that it can undergo agonist-dependent phosphorylation and sequestration. Here we demonstrate that stable expression of β2AR-GFP in HeLa cells enables a detailed temporal and spatial analysis of β2AR trafficking events associated with receptor sequestration, down-regulation, and recycling. Colocalization of the β2AR-GFP with rhodamine-labeled transferrin and rhodamine-labeled dextran during agonist treatment revealed sequential localization of receptor in cellular compartments containing these compounds. Moreover, this system appears capable of distinguishing the differing effects of various β-agonists (including the highly hydrophobic ligand salmeterol) on β2AR trafficking, overcoming some of the limitations in previous analyses of these compounds. Upon agonist stimulation, β2-adrenergic receptors (β2ARs) 1The abbreviations used are: β2AR, β2-adrenergic receptor; GFP, green fluorescent protein; PCR, polymerase chain reaction; PBS, phosphate-buffered saline. 1The abbreviations used are: β2AR, β2-adrenergic receptor; GFP, green fluorescent protein; PCR, polymerase chain reaction; PBS, phosphate-buffered saline. are rapidly desensitized by receptor phosphorylation (1Sterne-Marr R. Benovic J.L. Litwack G. Vitamins and Hormones. Academic Press, New York1995: 193-234Google Scholar). Receptor phosphorylation by G protein-coupled receptor kinases and subsequent binding of non-visual arrestins initiates the internalization of β2ARs via clathrin-coated pits (2Ferguson S.S.G. Downey III, W.E. Colapietro A.M. Barak L.S. Menard L. Caron M.G. Science. 1996; 271: 363-365Crossref PubMed Scopus (839) Google Scholar, 3Goodman O.B.J. Krupnick J.G. Santini F. Gurevich V.V. Penn R.B. Gagnon A.W. Keen J.H. Benovic J.L. Nature. 1996; 383: 447-450Crossref PubMed Scopus (1154) Google Scholar). Previous studies have demonstrated that internalized receptors have multiple potential fates. One such fate is the recycling of internalized receptors to the plasma membrane, presumably completing an ill defined “resensitization ” process involving β2AR dephosphorylation in an endosomal compartment (4Pippig S. Andexinger S. Lohse M. Mol. Pharmacol. 1995; 47: 666-676PubMed Google Scholar, 5Krueger K.M. Daaka Y. Pitcher J.A. Lefkowitz R.J. J. Biol. Chem. 1997; 272: 5-8Abstract Full Text Full Text PDF PubMed Scopus (312) Google Scholar). Depending upon the duration of agonist exposure, internalized β2ARs may ultimately appear “lost” or destroyed (undetectable by radioligand binding), ostensibly trafficking to lysosomes where they are degraded. Previously, the analysis of these events has been heavily dependent on biochemical and pharmacological approaches. Agonist-mediated subcellular redistribution of β2ARs was initially inferred from ligand binding studies (6Toews M.L. Liang M. Perkins J.P. Mol. Pharmacol. 1987; 32: 737-742PubMed Google Scholar, 7Kurz J.B. Perkins J.P. Mol. Pharmacol. 1992; 41: 375-381PubMed Google Scholar) and the coincident migration of β2ARs with enzyme markers (8Chuang D.M. Biochem. Biophys. Res. Commun. 1982; 105: 1466-1472Crossref PubMed Scopus (13) Google Scholar, 9Waldo G.L. Northup J.K. Perkins J.P. Harden T.K. J. Biol. Chem. 1983; 258: 13900-13908Abstract Full Text PDF PubMed Google Scholar) or epidermal growth factor (10Wakshull E. Hertel C. O'Keefe E.J. Perkins J.P. J. Cell. Biochem. 1985; 29: 127-141Crossref PubMed Scopus (10) Google Scholar, 11Liao J.-F. Perkins J.P. Mol. Pharmacol. 1993; 44: 364-370PubMed Google Scholar) into subcellular fractions resolved through centrifugation. However, direct visualization of β2AR trafficking events remained lacking until von Zastrow and Kobilka (12von Zastrow M. Kobilka B.K. J. Biol. Chem. 1992; 267: 3530-3538Abstract Full Text PDF PubMed Google Scholar) provided immunocytochemical evidence of rapid, agonist-induced redistribution of epitope-tagged β2ARs into small, punctate accumulations within the cytoplasm. The time course of β2AR redistribution assessed by confocal microscopy paralleled that of β2AR sequestration measured by radioligand binding. Importantly, internalized β2ARs colocalized with transferrin receptors, suggesting that sequestered β2ARs undergo processing through endosomal compartments in a manner similar to that observed for constitutively internalized receptors. Advances in the development of proteins conjugated with green fluorescent protein (GFP) have since provided the opportunity for real time optical analysis of protein trafficking events in individual cells (13Prasher D.C. Eckenrode V.K. Ward W.W. Prendergast F.G. Cormier M.J. Gene (Amst.). 1992; 111: 229-233Crossref PubMed Scopus (1748) Google Scholar, 14Kain S.R. Adams M. Kondepudi A. Yang T.-T. Ward W.W. Kitts P. BioTechniques. 1995; 19: 650-654PubMed Google Scholar, 15Inouye S. Tsuji F.I. FEBS Lett. 1994; 341: 277-280Crossref PubMed Scopus (353) Google Scholar). Green fluorescent protein is a naturally occurring protein isolated from several different species of jellyfish (Aequoria) and sea cucumbers (Renilla). When expressed in cells, proteins conjugated with GFP may be visualized with routine fluorescent microscopy without the need to fix cells. Recently, Barak et al. (16Barak L.S. Ferguson S.S.G. Zhang J. Martenson C. Meyer T. Caron M.G. Mol. Pharmacol. 1997; 51: 177-184Crossref PubMed Scopus (201) Google Scholar) established the utility of a β2AR-GFP fusion protein in visualizing agonist-mediated β2AR internalization in HEK293 cells transiently expressing the construct. This study also demonstrated that β2AR-GFP is fully functional with regard to ligand binding and adenylyl cyclase stimulation and that it can undergo agonist-dependent phosphorylation and sequestration. Here we demonstrate that stable expression of β2AR-GFP in HeLa cells enables a detailed temporal and spatial analysis of β2AR trafficking events associated with receptor sequestration, down-regulation, and recycling. Colocalization of the β2AR-GFP with rhodamine-labeled transferrin and rhodamine-labeled dextran during agonist treatment revealed sequential localization of receptor in cellular compartments containing these compounds. Moreover, this system appears capable of distinguishing the differing effects of various β-agonists (including the highly hydrophobic ligand salmeterol) on β2AR trafficking, overcoming some of the limitations in previous analyses of these compounds. We thank Drs. J. Keen and C. Schmutte and members of the Benovic and Keen labs for helpful discussions, J. Dispoto and P. Hingorani for confocal microscopy, and H. Alder and the Kimmel Nucleic Acids facility for DNA synthesis and sequencing. We also thank Dr. B. Kobilka for the Flag tagged β2AR construct, Dr. R. Lefkowitz for CHW cells, Dr. M. Ascoli for the cAMP antibody, Dr. P. Garcia for the GPT-Ad5, and Sepracor Inc. for providing formoterol, albuterol, and salmeterol.