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• W2108358725 abstract "Calcitonin receptor-like receptor (CLR) and the receptor activity-modifying protein 1 (RAMP1) comprise a receptor for calcitonin gene-related peptide (CGRP). Although CGRP induces endocytosis of CLR/RAMP1, little is known about post-endocytic sorting of these proteins. We observed that the duration of stimulation with CGRP markedly affected post-endocytic sorting of CLR/RAMP1. In HEK and SK-N-MC cells, transient stimulation (10-7 m CGRP, 1 h), induced CLR/RAMP1 recycling with similar kinetics (2-6 h), demonstrated by labeling receptors in living cells with antibodies to extracellular epitopes. Recycling of CLR/RAMP1 correlated with resensitization of CGRP-induced increases in [Ca2+]i. Cycloheximide did not affect resensitization, but bafilomycin A1, an inhibitor of vacuolar H+-ATPases, abolished resensitization. Recycling CLR and RAMP1 were detected in endosomes containing Rab4a and Rab11a, and expression of GTPase-defective Rab4aS22N and Rab11aS25N inhibited resensitization. After sustained stimulation (10-7 m CGRP, >2 h), CLR/RAMP1 trafficked to lysosomes. RAMP1 was degraded ∼4-fold more rapidly than CLR (RAMP1, 45% degradation, 5 h; CLR, 54% degradation, 16 h), determined by Western blotting. Inhibitors of lysosomal, but not proteasomal, proteases prevented degradation. Sustained stimulation did not induce detectable mono- or polyubiquitination of CLR or RAMP1, determined by immunoprecipitation and Western blotting. Moreover, a RAMP1 mutant lacking the only intracellular lysine (RAMP1K142R) internalized and was degraded normally. Thus, after transient stimulation with CGRP, CLR and RAMP1 traffic from endosomes to the plasma membrane, which mediates resensitization. After sustained stimulation, CLR and RAMP1 traffic from endosomes to lysosomes by ubiquitin-independent mechanisms, where they are degraded at different rates. Calcitonin receptor-like receptor (CLR) and the receptor activity-modifying protein 1 (RAMP1) comprise a receptor for calcitonin gene-related peptide (CGRP). Although CGRP induces endocytosis of CLR/RAMP1, little is known about post-endocytic sorting of these proteins. We observed that the duration of stimulation with CGRP markedly affected post-endocytic sorting of CLR/RAMP1. In HEK and SK-N-MC cells, transient stimulation (10-7 m CGRP, 1 h), induced CLR/RAMP1 recycling with similar kinetics (2-6 h), demonstrated by labeling receptors in living cells with antibodies to extracellular epitopes. Recycling of CLR/RAMP1 correlated with resensitization of CGRP-induced increases in [Ca2+]i. Cycloheximide did not affect resensitization, but bafilomycin A1, an inhibitor of vacuolar H+-ATPases, abolished resensitization. Recycling CLR and RAMP1 were detected in endosomes containing Rab4a and Rab11a, and expression of GTPase-defective Rab4aS22N and Rab11aS25N inhibited resensitization. After sustained stimulation (10-7 m CGRP, >2 h), CLR/RAMP1 trafficked to lysosomes. RAMP1 was degraded ∼4-fold more rapidly than CLR (RAMP1, 45% degradation, 5 h; CLR, 54% degradation, 16 h), determined by Western blotting. Inhibitors of lysosomal, but not proteasomal, proteases prevented degradation. Sustained stimulation did not induce detectable mono- or polyubiquitination of CLR or RAMP1, determined by immunoprecipitation and Western blotting. Moreover, a RAMP1 mutant lacking the only intracellular lysine (RAMP1K142R) internalized and was degraded normally. Thus, after transient stimulation with CGRP, CLR and RAMP1 traffic from endosomes to the plasma membrane, which mediates resensitization. After sustained stimulation, CLR and RAMP1 traffic from endosomes to lysosomes by ubiquitin-independent mechanisms, where they are degraded at different rates. Calcitonin gene-related peptide (CGRP) 2The abbreviations used are: CGRP, calcitonin gene-related peptide; GPCR, G-protein-coupled receptor; CLR, calcitonin receptor-like receptor; RAMP1, receptor activity-modifying protein; NK1R, neurokinin 1 receptor; β2AR, β2 adrenergic receptor; PAR2, protease-activated receptor 2; LAMP1, lysosomal-associated glycomembrane protein 1; EEA1, early endosomal antigen 1; BSA, bovine serum albumin; HA, hemagglutinin; PBS, phosphate-buffered saline; GFP, green fluorescent protein.2The abbreviations used are: CGRP, calcitonin gene-related peptide; GPCR, G-protein-coupled receptor; CLR, calcitonin receptor-like receptor; RAMP1, receptor activity-modifying protein; NK1R, neurokinin 1 receptor; β2AR, β2 adrenergic receptor; PAR2, protease-activated receptor 2; LAMP1, lysosomal-associated glycomembrane protein 1; EEA1, early endosomal antigen 1; BSA, bovine serum albumin; HA, hemagglutinin; PBS, phosphate-buffered saline; GFP, green fluorescent protein. belongs to the calcitonin family of regulatory peptides and is produced by tissue-specific alternate splicing of transcripts from the calcitonin gene (1Amara S.G. Jonas V. Rosenfeld M.G. Ong E.S. Evans R.M. Nature. 1982; 298: 240-244Crossref PubMed Scopus (1738) Google Scholar). CGRP is a potent vasodilator and mediator of neurogenic inflammation and pain transmission (2van Rossum D. Hanisch U.K. Quirion R. Neurosci. Biobehav. Rev. 1997; 21: 649-678Crossref PubMed Scopus (442) Google Scholar, 3Brain S.D. Grant A.D. Physiol. Rev. 2004; 84: 903-934Crossref PubMed Scopus (616) Google Scholar). Notably, CGRP has a causative role in migraine headaches and is thus a mediator of human disease (4Olesen J. Diener H.C. Husstedt I.W. Goadsby P.J. Hall D. Meier U. Pollentier S. Lesko L.M. N. Engl. J. Med. 2004; 350: 1104-1110Crossref PubMed Scopus (1024) Google Scholar). In view of the importance of CGRP in health and disease, it is of interest to understand the mechanisms that control cellular responses to this peptide. Unusually for neuropeptides, the CGRP receptor is a heterodimer composed of calcitonin receptor-like receptor (CLR) and receptor activity-modifying protein 1 (RAMP1). CLR is a G-protein-coupled receptor (GPCR) that shares 55% amino acid sequence identity with the calcitonin receptor (5Njuki F. Nicholl C.G. Howard A. Mak J.C. Barnes P.J. Girgis S.I. Legon S. Clin. Sci. (Lond.). 1993; 85: 385-388Crossref PubMed Scopus (131) Google Scholar), whereas RAMP1, RAMP2, and RAMP3 are single transmembrane proteins with ∼30% identity (6McLatchie L.M. Fraser N.J. Main M.J. Wise A. Brown J. Thompson N. Solari R. Lee M.G. Foord S.M. Nature. 1998; 393: 333-339Crossref PubMed Scopus (1842) Google Scholar). CLR functions as either a CGRP receptor, when coexpressed with RAMP1, or an adrenomedullin receptor, when coexpressed with RAMP2 or -3 (6McLatchie L.M. Fraser N.J. Main M.J. Wise A. Brown J. Thompson N. Solari R. Lee M.G. Foord S.M. Nature. 1998; 393: 333-339Crossref PubMed Scopus (1842) Google Scholar); the extracellular domain of the RAMP imparts this specificity (7Kuwasako K. Kitamura K. Onitsuka H. Uemura T. Nagoshi Y. Kato J. Eto T. FEBS Lett. 2002; 519: 113-116Crossref PubMed Scopus (24) Google Scholar, 8Kuwasako K. Kitamura K. Nagoshi Y. Cao Y.N. Eto T. J. Biol. Chem. 2003; 278: 22623-22630Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). When expressed alone, CLR is retained in the endoplasmic reticulum (6McLatchie L.M. Fraser N.J. Main M.J. Wise A. Brown J. Thompson N. Solari R. Lee M.G. Foord S.M. Nature. 1998; 393: 333-339Crossref PubMed Scopus (1842) Google Scholar), and RAMP1 is retained in the endoplasmic reticulum and the Golgi apparatus (9Fraser N.J. Wise A. Brown J. McLatchie L.M. Main M.J. Foord S.M. Mol. Pharmacol. 1999; 55: 1054-1059Crossref PubMed Scopus (167) Google Scholar, 10Christopoulos G. Perry K.J. Morfis M. Tilakaratne N. Gao Y. Fraser N.J. Main M.J. Foord S.M. Sexton P.M. Mol. Pharmacol. 1999; 56: 235-242Crossref PubMed Scopus (412) Google Scholar, 11Kuwasako K. Shimekake Y. Masuda M. Nakahara K. Yoshida T. Kitaura M. Kitamura K. Eto T. Sakata T. J. Biol. Chem. 2000; 275: 29602-29609Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar). However, when coexpressed, CLR and RAMP1 traffic to the plasma membrane. Thus, RAMP1 is also a chaperone that targets CLR to the plasma membrane. However, it remains to be determined if CLR and RAMP1 are invariably associated after receptor activation. Upon activation with CGRP, CLR, but not RAMP1, is phosphorylated and interacts with β-arrestins (12Hilairet S. Belanger C. Bertrand J. Laperriere A. Foord S.M. Bouvier M. J. Biol. Chem. 2001; 276: 42182-42190Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar). β-Arrestins are adapters for clathrin and AP2, and the CLR, RAMP1, and β-arrestin complex undergoes dynamin-dependent endocytosis in clathrin-coated pits by well defined mechanisms (11Kuwasako K. Shimekake Y. Masuda M. Nakahara K. Yoshida T. Kitaura M. Kitamura K. Eto T. Sakata T. J. Biol. Chem. 2000; 275: 29602-29609Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar, 12Hilairet S. Belanger C. Bertrand J. Laperriere A. Foord S.M. Bouvier M. J. Biol. Chem. 2001; 276: 42182-42190Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar). However, little is known about the mechanisms of post-endocytic sorting of GPCRs, such as CLR, or accessory proteins, such as RAMP1, to degradative or recycling pathways. It is important to understand the post-endocytic sorting of CLR and RAMP1, because recycling may permit rapid resensitization of CGRP signaling, whereas degradation would prevent sustained, uncontrolled CGRP signaling during conditions of sustained peptide release. β-Arrestins can influence the rate of recycling of GPCRs. “Class A” receptors (e.g. β2 adrenergic receptor (β2AR), μ-opioid receptor, neurokinin 3 receptor) form low affinity and transient interactions with β-arrestin2 and rapidly recycle (13Oakley R.H. Laporte S.A. Holt J.A. Caron M.G. Barak L.S. J. Biol. Chem. 2000; 275: 17201-17210Abstract Full Text Full Text PDF PubMed Scopus (679) Google Scholar, 14Schmidlin F. Roosterman D. Bunnett N.W. Am. J. Physiol. Cell Physiol. 2003; 285: C945-C958Crossref PubMed Scopus (36) Google Scholar). “Class B” receptors (e.g. neurokinin 1 receptor (NK1R)) form high affinity and sustained interactions with β-arrestin1 and -2 and slowly recycle (13Oakley R.H. Laporte S.A. Holt J.A. Caron M.G. Barak L.S. J. Biol. Chem. 2000; 275: 17201-17210Abstract Full Text Full Text PDF PubMed Scopus (679) Google Scholar, 14Schmidlin F. Roosterman D. Bunnett N.W. Am. J. Physiol. Cell Physiol. 2003; 285: C945-C958Crossref PubMed Scopus (36) Google Scholar). Activated CLR colocalizes with β-arrestin2 and β-arrestin1 for prolonged periods (12Hilairet S. Belanger C. Bertrand J. Laperriere A. Foord S.M. Bouvier M. J. Biol. Chem. 2001; 276: 42182-42190Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar). 3N. W. Bunnett, unpublished observation.3N. W. Bunnett, unpublished observation. Thus, CLR may belong to the class B family of slowly recycling GPCRs. However, it is not known whether CLR and RAMP1 can recycle. Addition of ubiquitin molecules to intracellular lysine residues targets some GPCRs to degradative pathways (e.g. β2AR, chemokine (CXC motif) receptor 4, protease-activated receptor 2 (PAR2), and NK1R) (15Shenoy S.K. McDonald P.H. Kohout T.A. Lefkowitz R.J. Science. 2001; 294: 1307-1313Crossref PubMed Scopus (706) Google Scholar, 16Marchese A. Benovic J.L. J. Biol. Chem. 2001; 276: 45509-45512Abstract Full Text Full Text PDF PubMed Scopus (390) Google Scholar, 17Marchese A. Raiborg C. Santini F. Keen J.H. Stenmark H. Benovic J.L. Dev. Cell. 2003; 5: 709-722Abstract Full Text Full Text PDF PubMed Scopus (314) Google Scholar, 18Jacob C. Cottrell G.S. Gehringer D. Schmidlin F. Grady E.F. Bunnett N.W. J. Biol. Chem. 2005; 280: 16076-16087Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar, 19Cottrell G.S. Padilla B. Pikios S. Roosterman D. Steinhoff M. Gehringer D. Grady E.F. Bunnett N.W. J. Biol. Chem. 2006; 281: 27773-27783Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). However, other GPCRs traffic to lysosomes by ubiquitin-independent processes (e.g. δ-opioid receptor) (20Tanowitz M. Von Zastrow M. J. Biol. Chem. 2002; 277: 50219-50222Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). The nature of the stimulus also affects ubiquitination and post-endocytic sorting of GPCRs. Thus, after transient stimulation with low concentrations of substance P, the NK1R is not ubiquitinated and rapidly recycles and resensitizes (19Cottrell G.S. Padilla B. Pikios S. Roosterman D. Steinhoff M. Gehringer D. Grady E.F. Bunnett N.W. J. Biol. Chem. 2006; 281: 27773-27783Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 21Grady E.F. Garland A.M. Gamp P.D. Lovett M. Payan D.G. Bunnett N.W. Mol. Biol. Cell. 1995; 6: 509-524Crossref PubMed Scopus (201) Google Scholar, 22Garland A.M. Grady E.F. Lovett M. Vigna S.R. Frucht M.M. Krause J.E. Bunnett N.W. Mol. Pharmacol. 1996; 49: 438-446PubMed Google Scholar, 23Roosterman D. Cottrell G.S. Schmidlin F. Steinhoff M. Bunnett N.W. J. Biol. Chem. 2004; 279: 30670-30679Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). In contrast, after sustained stimulation with high concentrations of substance P, the NK1R is ubiquitinated and degraded (19Cottrell G.S. Padilla B. Pikios S. Roosterman D. Steinhoff M. Gehringer D. Grady E.F. Bunnett N.W. J. Biol. Chem. 2006; 281: 27773-27783Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). Although CLR and RAMP1 exist in a stoichiometric 1:1 ratio at the plasma membrane (12Hilairet S. Belanger C. Bertrand J. Laperriere A. Foord S.M. Bouvier M. J. Biol. Chem. 2001; 276: 42182-42190Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar), and co-internalize and traffic to lysosomes (11Kuwasako K. Shimekake Y. Masuda M. Nakahara K. Yoshida T. Kitaura M. Kitamura K. Eto T. Sakata T. J. Biol. Chem. 2000; 275: 29602-29609Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar, 12Hilairet S. Belanger C. Bertrand J. Laperriere A. Foord S.M. Bouvier M. J. Biol. Chem. 2001; 276: 42182-42190Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar), their respective rates of degradation have not been examined, and the role of ubiquitination in lysosomal trafficking of CLR and RAMP1 is unknown. It remains to be determined if the duration of stimulation with CGRP affects the post-endocytic sorting of CLR and RAMP1 to recycling or degradative pathways. We investigated the pathway and mechanism of post-endocytic trafficking of CLR and RAMP1 and determined the importance of this trafficking to the control of CGRP signaling. Our aims were as follows: (a) to determine whether transient stimulation with CGRP induces post-endocytic sorting of CLR and RAMP1 to recycling pathways; (b) to define the role of recycling in resensitization of CGRP signaling; (c) to determine whether sustained stimulation with CGRP induces post-endocytic sorting of CLR and RAMP1 to degradative pathways; and (d) to investigate whether trafficking to degradative pathways involves ubiquitination of CLR and RAMP1. Reagents—Rabbit antibodies to the C terminus of rat CLR (RK11) and human RAMP1 (9891) have been described previously (24Cottrell G.S. Roosterman D. Marvizon J.C. Song B. Wick E. Pikios S. Wong H. Berthelier C. Tang Y. Sternini C. Bunnett N.W. Grady E.F. J. Comp. Neurol. 2005; 490: 239-255Crossref PubMed Scopus (90) Google Scholar). Sources of other antibodies were: rat high affinity anti-HA (Roche Applied Science); rabbit anti-Myc and mouse anti-β-actin (A-5441, Sigma); goat anti-Myc (A-14) and mouse anti-ubiquitin (P4D1) (Santa Cruz Biotechnology, Santa Cruz, CA); mouse anti-human lysosomal-associated glycomembrane protein 1 (LAMP1, Developmental Studies Hybridoma Bank, Iowa City, IA); mouse anti-early endosomal antigen 1 (EEA1, BD Transduction Laboratories); goat or donkey anti-mouse, rat, or rabbit IgG coupled to horseradish peroxidase, fluorescein isothiocyanate, rhodamine red-X, or Cy5 (Jackson ImmunoResearch, West Grove, PA); and goat anti-mouse or rabbit IgG coupled to AlexaFluor® 680 (Invitrogen) and coupled to IRDye™ 800 (Rockland Immunochemicals, Gilbertsville, PA). Rat α-CGRP was from Bachem (Torrance, CA). Vector Construction—cDNAs encoding rat CLR with an extracellular, N-terminal HA epitope, and rat RAMP1 with an extracellular, N-terminal Myc epitope have been described before (24Cottrell G.S. Roosterman D. Marvizon J.C. Song B. Wick E. Pikios S. Wong H. Berthelier C. Tang Y. Sternini C. Bunnett N.W. Grady E.F. J. Comp. Neurol. 2005; 490: 239-255Crossref PubMed Scopus (90) Google Scholar). CLR was subcloned into pcDNA5/FRT to yield pcDNA5/FRT-rCLR. To create a vector that expressed CLR and RAMP1, RAMP1 was amplified by PCR together with a cytomegalovirus promoter and bovine growth hormone poly(A) tail and subcloned into the BsmI and BstZ17I sites of pcDNA5/FRT-rCLR. A rat RAMP1 mutant in which the lysine (Lys-142) was mutated to arginine (designated RAMP1K142R) was generated by PCR (forward primer, 5′-cgcaaatgggcggtaggcgtg-3′; reverse primer, 5′-atgcggccgcctacacgatgccctctgtgcgcctgctcctcc-3′) using standard techniques and subcloned into pcDNA5/FRT-rCLR. Constructs were sequenced to verify integrity. Other primer sequences are available on request. cDNAs encoding GFP-tagged Rab4a, Rab11a, and GTPase-defective Rab4aS22N and Rab11aS25N have been described (23Roosterman D. Cottrell G.S. Schmidlin F. Steinhoff M. Bunnett N.W. J. Biol. Chem. 2004; 279: 30670-30679Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). Transfected Cells and Cell Lines—The generation and maintenance of human embryonic kidney 293 (HEK) FLP cells (Invitrogen) stably expressing rat CLR and rat RAMP1 have been described (24Cottrell G.S. Roosterman D. Marvizon J.C. Song B. Wick E. Pikios S. Wong H. Berthelier C. Tang Y. Sternini C. Bunnett N.W. Grady E.F. J. Comp. Neurol. 2005; 490: 239-255Crossref PubMed Scopus (90) Google Scholar). HEKFLP cells stably expressing CLR and RAMP1 from the same vector (pcDNA5/FRT) or rat NK1R were created with the Flp-In™ system according to the manufacturer's guidelines, and cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated fetal bovine serum and 100 μg/ml hygromycin B. In some experiments, cells were transiently transfected with CLR, RAMP1, Rab4a, Rab4aS22N, Rab11a, or Rab11aS25N using Lipofectamine™ 2000 (Invitrogen) according to the manufacturer's guidelines. The human neuroblastoma cell line SK-N-MC was from American Tissue Type Collection (Manassas, VA). SK-N-MC cells were transiently transfected with CLR and RAMP1 and grown in minimal essential medium supplemented with non-essential amino acids and 10% HIFBS. All cells were routinely grown in 95% air, 5% CO2 at 37 °C. In control experiments, cells were transfected with vectors without inserts. Measurement of [Ca2+]i—Cells were incubated with 2.5 μm fura-2AM (Invitrogen) for 20 min at 37 °C and washed. Fluorescence was measured at 340 and 380 nm excitation and 510 nm emission in an F-2500 spectrophotometer (Hitachi Instruments, Irvine, CA). The ratio of the fluorescence at the two excitation wavelengths, which is proportional to [Ca2+]i, was calculated, and results are expressed as increase above basal values. To assess desensitization and resensitization, cells were challenged with CGRP (10-7 m, 1 h) or vehicle (control) and washed, and [Ca2+] was determined after a second challenge with CGRP (3 × 10-8 m). Immunofluorescence—Cells were plated at 3 × 105 cells per 35-mm dish, onto coverslips were coated with poly-d-lysine (100 μg/ml). To localize CLR and RAMP1, cells were fixed in 4% paraformaldehyde in 100 mm PBS, pH 7.4 (20 min at 4 °C), and washed for 15 min with 1× PBS containing 0.1% saponin and 1% normal goat serum or 2% normal donkey serum. Proteins were localized using primary antibodies: CLR (RK11, 1:4000), RAMP1 (9891, 1:2000 or goat anti-Myc, 1:100), LAMP1 (1:1000), and EEA1 (1:500) (overnight, 4 °C). Cells were washed for 15 min with 1× PBS containing 0.1% saponin and 1% normal goat serum or 2% normal donkey serum and incubated with secondary antibodies conjugated to fluorescein isothiocyanate, rhodamine red-X, or Cy5 (1:200, 2 h at room temperature or overnight at 4 °C). Trafficking of Antibody-tagged Receptors—To label CLR and RAMP1 at the cell surface, living cells were incubated with rat anti-HA (to detect CLR; 1:100) and rabbit or goat anti-Myc (to detect RAMP1; 1:100) for 30 min at 37 °C. Cells were washed with PBS containing Ca2+ and Mg2+ and stimulated with CGRP. Cells were fixed at specified times, washed, and incubated with fluorescent secondary antibodies overnight at 4 °C. Confocal Microscopy—Cells were observed by using Zeiss Axiovert, 510Meta, and Bio-Rad MRC1000 confocal microscopes with a Plan Apo ×100 (numerical aperture 1.3 or 1.4) objective. Images were collected at zooms of 1-2 and an iris of <3 μm, and typically 5-10 optical sections were taken at intervals of 0.5 μm. Images (single optical sections are shown) were colored and processed to adjust contrast and brightness using Adobe Photoshop CS (Adobe Systems, Mountain View, CA). SDS-PAGE and Western Blotting—Cells were lysed in 50 mm Tris/HCl, pH 7.4, 1% SDS, boiled, and centrifuged. Lysates (5-10 μg of protein) were separated by SDS-PAGE (CLR, 8 or 9%; RAMP1, 15% acrylamide gels). Proteins were transferred to polyvinylidene difluoride membranes (Immobilon-P or FL, Millipore, Billerica, MA) and blocked for 1 h at room temperature (1x PBS, 2% bovine serum albumin, 5% milk powder, 0.1% Tween 20, or Odyssey Blocking Buffer). Membranes were incubated with antibodies to rat CLR (RK11, 1:10,000), rat RAMP1 (9891, 1:5,000), β-actin (1:20,000), or ubiquitin P4D1 (1:1,000) overnight at 4 °C (1× PBS, 2% bovine serum albumin, 5% milk powder, 0.1% Tween 20, or Odyssey Blocking Buffer). Membranes were washed for 30 min (1× PBS, 0.1% Tween 20) and incubated with secondary antibody coupled to horseradish peroxidase (1:10,000, 1 h, room temperature). Membranes were washed for 30 min, and immunoreactive proteins were detected by using chemiluminescence (SuperSignal West Pico Chemiluminescent Substrate, Pierce). Alternatively, membranes were incubated with secondary antibodies conjugated to AlexaFluor® 680 or IRDye™ 800 (1:10-20,000, 1 h, room temperature), and blots were analyzed with the Odyssey infrared imaging system (Li-COR Biosciences, Lincoln, NE). Immunoprecipitation—Cells were plated at 1.5 × 106 cells per 100-mm dish and used after 48 h. After treatments, cells were washed with PBS containing Ca2+ and Mg2+, lysed with 1 ml of radioimmune precipitation assay buffer (10 mm Tris/HCl, pH 7.4, 150 mm NaCl, 50 mm NaF, 1 mm Na3VO4, 0.5% w/v sodium deoxycholate, 1% Nonidet P-40), and centrifuged (10 min, 15,000 g, room temperature). Supernatants were transferred to fresh tubes, and immunoprecipitating antibodies were added (CLR: rat anti-HA, 500 ng; RAMP1: rabbit anti-Myc, 2 μg). Samples were rotated for 16 h at 4 °C. Protein A/G Plus (Santa Cruz Biotechnology) was added (30 μl), and samples were rotated for 2 h at 4 °C. Immunoprecipitates were pelleted, washed three times with 1 ml of radioimmune precipitation assay, boiled in 2× Laemmli buffer, and analyzed by Western blotting. For denaturing immunoprecipitation (NK1R), cells were lysed in 100 μl of 10 mm Tris/HCl, pH 7.4, 1% SDS, sonicated, and mixed with 4 volumes of radioimmune precipitation assay buffer (10 mm Tris/HCl, pH 7.4, 150 mm NaCl, 50 mm NaF, 1 mm Na3VO4, 0.5% w/v sodium deoxycholate, 1% Nonidet P-40). The lysate was mixed by pipetting, and a radioimmune precipitation assay was added to 1-ml final volume and centrifuged (16,000 × g, 20 min, 4 °C). Supernatants were rotated with antibody (FLAG M2, 3.5 μg/ml) overnight at 4 °C, and samples were processed as described above. Activation of CLR and RAMP1 and Drug Treatments—Two days after plating cells or after transfection, cells were washed three times with PBS containing Ca2+ and Mg2+ and placed in Dulbecco's modified Eagle's medium containing 0.1% bovine serum albumin. Cells were stimulated with 10-7 m rat α-CGRP, unless otherwise stated. To inhibit new protein synthesis, cells were treated with cycloheximide (140 μm, Sigma). To inhibit vacuolar-type H+-ATPases, cells were treated with bafilomycin A1 (1 μm, A. G. Scientific, San Diego, CA). These inhibitors were preincubated with cells 1 h prior to stimulation with CGRP and were present throughout stimulation and recovery phases of the experiments. Lysosomal proteases were inhibited using ZPAD, E64d (Bachem, 200 μm and 20 μm, respectively) and pepstatin A (Roche Applied Science, 10 μm). The proteolytic activity of the proteasome was inhibited using epoxomicin (Biomol, Plymouth Meeting, PA; 10 μm). Controls included appropriate vehicle. Densitometry—Signals on Western blots were quantified using the Odyssey Infrared Imaging System (Li-COR Biosciences), or blots were digitized using an Epson Perfection 3200 PHOTO scanner and analyzed by densitometry with National Institutes of Health Image 1.63. To quantify CLR and RAMP1 degradation, signals were compared with β-actin signals. Statistics—Data are presented as the mean and standard error of ≥ 3 experiments. Results are compared by Student's t test or analysis of variance and Student-Newman-Kuel's test, with *, p < 0.05 considered significant. CLR and RAMP1 Traffic to Early Endosomes and Lysosomes—To examine the effects of CGRP on trafficking of CLR and RAMP1, we incubated HEK cells stably expressing CLR and RAMP1 (HEK-CLR-RAMP1 cells) with CGRP (10-7 m) for 0-120 min; we localized CLR, RAMP1 (using antibodies RK11 and 9891, to the C terminus of CLR and RAMP1, respectively), EEA1 and LAMP1 by indirect immunofluorescence. In unstimulated cells, CLR and RAMP1 were at the plasma membrane (Fig. 1, A and B, arrowheads). After 10 min with CGRP, CLR and RAMP1 were depleted from the plasma membrane and were colocalized with EEA1 in early endosomes (Fig. 1, A and B, white arrows). CLR and RAMP1 were prominently localized to early endosomes at 30 and 60 min (not shown). After 120 min with CGRP, CLR and RAMP1 were sometimes colocalized with EEA1 (Fig. 1, A and B, white arrows) but were also detected in vesicles that did not contain EEA1 (Fig. 1, A and B, yellow arrows). These vesicles were lysosomes, because at 120 min CLR and RAMP1 were prominently colocalized with LAMP1 (Fig. 1, C and D, white arrows). However, CLR and RAMP1 were also detected in some vesicles that did not contain LAMP1 and are probably early endosomes (Fig. 1, C and D, yellow arrows). At earlier times (e.g. 60 min), CLR and LAMP1 were rarely detected in lysosomes (not shown). These results show that activated CLR and RAMP1 traffic from the plasma membrane to early endosomes and lysosomes, which is consistent with other reports of CLR and RAMP1 trafficking (11Kuwasako K. Shimekake Y. Masuda M. Nakahara K. Yoshida T. Kitaura M. Kitamura K. Eto T. Sakata T. J. Biol. Chem. 2000; 275: 29602-29609Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar). At early times (10-60 min), the receptors are most prominently in early endosomes. At later times (120 min), the receptors are more prominently sorted to lysosomes. However, even after sustained activation, lysosomal sorting is incomplete and some receptors are retained in early endosomes. We therefore examined whether CLR and RAMP1 can recycle from early endosomes after transient stimulation with CGRP. CLR and RAMP1 Recycle after Transient Stimulation with CGRP—Although continuous stimulation of cells with high concentrations of CGRP induces trafficking of CLR and RAMP1 to lysosomes (11Kuwasako K. Shimekake Y. Masuda M. Nakahara K. Yoshida T. Kitaura M. Kitamura K. Eto T. Sakata T. J. Biol. Chem. 2000; 275: 29602-29609Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar), many GPCRs efficiently recycle, especially after brief stimulation with agonist (22Garland A.M. Grady E.F. Lovett M. Vigna S.R. Frucht M.M. Krause J.E. Bunnett N.W. Mol. Pharmacol. 1996; 49: 438-446PubMed Google Scholar, 23Roosterman D. Cottrell G.S. Schmidlin F. Steinhoff M. Bunnett N.W. J. Biol. Chem. 2004; 279: 30670-30679Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). We examined CLR and RAMP1 recycling in cycloheximide-treated HEK-CLR-RAMP1 cells, to avoid synthesis of new receptors, which would also traffic to the plasma membrane. Cells were incubated with CGRP for 1 h to induce trafficking of both proteins to early endosomes. Cells were washed and incubated in CGRP-free medium for 0-6 h, to allow recycling. CLR and RAMP1 were simultaneously detected by indirect immunofluorescence using a rabbit antibody the C terminus of CLR (RK11), and a goat antibody to an N-terminal Myc epitope of RAMP1. In unstimulated cells, CLR and RAMP1 colocalized at the plasma membrane (Fig. 2A, arrowheads). After 1 h with CGRP, CLR and RAMP1 were depleted from the plasma membrane and were colocalized in vesicles (Fig. 2A, arrows). These vesicles colocalized with EEA1 and are thus early endosomes (not shown). At 2 h after washing and recovery in agonist-free medium, CLR and RAMP1 were present in some endosomes but were also detected at the plasma membrane, and both proteins were almost exclusively colocalized at the plasma membrane after 6-h recovery (Fig. 2A, arrowheads). The reappearance of CLR and RAMP1 at the plasma membrane of cycloheximide-treated cells suggests that these proteins can recycle. However, mobilization of CLR and RAMP1 from intracellular stores could also account for the reappearance of CLR and RAMP1 at the plasma membrane. To exclude this possibility, we examined trafficking of CLR and RAMP1 that were tagged at the cell surface using antibodies to extracellular epitopes. To label surface receptors, living HEK-CLR-RAMP1 cells were simultaneously incubated with antibodies to the extracellular epitop" @default.
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• W2108358725 title "Post-endocytic Sorting of Calcitonin Receptor-like Receptor and Receptor Activity-modifying Protein 1" @default.
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