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Novel Function for Receptor Activity-modifying Proteins (RAMPs) in Post-endocytic Receptor Trafficking

2004; Elsevier BV; Volume: 280; Issue: 10 Linguagem: Inglês

10.1074/jbc.m413786200

1083-351X

Jennifer M. Bomberger, Narayanan Parameswaran, Carolyn Hall, Nambi Aiyar, W. S. Spielman,

Pancreatic function and diabetes

RAMPs (1McLatchie 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 (1868) Google Scholar–3Kamitani S. Asakawa M. Shimekake Y. Kuwasako K. Nakahara K. Sakata T. FEBS Lett. 1999; 448: 111-114Crossref PubMed Scopus (112) Google Scholar) are single transmembrane accessory proteins crucial for plasma membrane expression, which also determine receptor phenotype of various G-protein-coupled receptors. For example, adrenomedullin receptors are comprised of RAMP2 or RAMP3 (AM1R and AM2R, respectively) and calcitonin receptor-like receptor (CRLR), while a CRLR heterodimer with RAMP1 yields a calcitonin gene-related peptide receptor. The major aim of this study was to determine the role of RAMPs in receptor trafficking. We hypothesized that a PDZ type I domain present in the C terminus of RAMP3, but not in RAMP1 or RAMP2, leads to protein-protein interactions that determine receptor trafficking. Employing adenylate cyclase assays, radioligand binding, and immunofluorescence microscopy, we observed that in HEK293 cells the CRLR-RAMP complex undergoes agonist-stimulated desensitization and internalization and fails to resensitize (i.e. degradation of the receptor complex). Co-expression of N-ethylmaleimide-sensitive factor (NSF) with the CRLR-RAMP3 complex, but not CRLR-RAMP1 or CRLR-RAMP2 complex, altered receptor trafficking to a recycling pathway. Mutational analysis of RAMP3, by deletion and point mutations, indicated that the PDZ motif of RAMP3 interacts with NSF to cause the change in trafficking. The role of RAMP3 and NSF in AM2R recycling was confirmed in rat mesangial cells, where RNA interference with RAMP3 and pharmacological inhibition of NSF both resulted in a lack of receptor resensitization/recycling after agonist-stimulated desensitization. These findings provide the first functional difference between the AM1R and AM2R at the level of post-endocytic receptor trafficking. These results indicate a novel function for RAMP3 in the post-endocytic sorting of the AM-R and suggest a broader regulatory role for RAMPs in receptor trafficking. RAMPs (1McLatchie 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 (1868) Google Scholar–3Kamitani S. Asakawa M. Shimekake Y. Kuwasako K. Nakahara K. Sakata T. FEBS Lett. 1999; 448: 111-114Crossref PubMed Scopus (112) Google Scholar) are single transmembrane accessory proteins crucial for plasma membrane expression, which also determine receptor phenotype of various G-protein-coupled receptors. For example, adrenomedullin receptors are comprised of RAMP2 or RAMP3 (AM1R and AM2R, respectively) and calcitonin receptor-like receptor (CRLR), while a CRLR heterodimer with RAMP1 yields a calcitonin gene-related peptide receptor. The major aim of this study was to determine the role of RAMPs in receptor trafficking. We hypothesized that a PDZ type I domain present in the C terminus of RAMP3, but not in RAMP1 or RAMP2, leads to protein-protein interactions that determine receptor trafficking. Employing adenylate cyclase assays, radioligand binding, and immunofluorescence microscopy, we observed that in HEK293 cells the CRLR-RAMP complex undergoes agonist-stimulated desensitization and internalization and fails to resensitize (i.e. degradation of the receptor complex). Co-expression of N-ethylmaleimide-sensitive factor (NSF) with the CRLR-RAMP3 complex, but not CRLR-RAMP1 or CRLR-RAMP2 complex, altered receptor trafficking to a recycling pathway. Mutational analysis of RAMP3, by deletion and point mutations, indicated that the PDZ motif of RAMP3 interacts with NSF to cause the change in trafficking. The role of RAMP3 and NSF in AM2R recycling was confirmed in rat mesangial cells, where RNA interference with RAMP3 and pharmacological inhibition of NSF both resulted in a lack of receptor resensitization/recycling after agonist-stimulated desensitization. These findings provide the first functional difference between the AM1R and AM2R at the level of post-endocytic receptor trafficking. These results indicate a novel function for RAMP3 in the post-endocytic sorting of the AM-R and suggest a broader regulatory role for RAMPs in receptor trafficking. The recent discovery of receptor activity-modifying proteins (RAMPs) 1The abbreviations used are: RAMP, receptor activity-modifying protein; GPCR, G-protein-coupled receptor; CRLR, calcitonin receptor-like receptor; CGRP, calcitonin gene-related peptide; β2-AR, β2-adrenergic receptor; NSF, N-ethylmaleimide-sensitive factor; AMPA, α-amino-3-hydroxy-5-methylisoxazolepropionate; SNARE, soluble NSF attachment protein receptor; FBS, fetal bovine serum; DMEM, Dulbecco's modified Eagle's medium; BSA, bovine serum albumin; dPBS, Dulbecco's phosphate-buffered saline; d-siRNA, diced small interfering RNA; RMC, rat mesangial cell; GST, glutathione S-transferase; NEM, N-ethylmaleimide; AM (or ADM), adrenomedullin; AC, adenylate cyclase; GFP, green fluorescent protein; EGFP, enhanced GFP. has raised new possibilities for modes of regulation of G-protein-coupled receptors (GPCRs). RAMPs were discovered as accessory proteins indispensable to the function of an orphan GPCR, now termed the calcitonin receptor-like receptor (CRLR) (1McLatchie 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 (1868) Google Scholar). Three RAMP isoforms (1McLatchie 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 (1868) Google Scholar, 2Kuwasako 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 (145) Google Scholar, 3Kamitani S. Asakawa M. Shimekake Y. Kuwasako K. Nakahara K. Sakata T. FEBS Lett. 1999; 448: 111-114Crossref PubMed Scopus (112) Google Scholar) have been identified as distinct gene products that yield single transmembrane-spanning proteins. RAMPs are required for the plasma membrane expression, as well as for determination of receptor phenotype for CRLR (selective ligand recognition) (1McLatchie 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 (1868) Google Scholar, 2Kuwasako 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 (145) Google Scholar). Co-expression of RAMP1 with CRLR yields a calcitonin gene-related peptide-1 (CGRP-1) receptor, while coexpression of RAMP2 or RAMP3 with CRLR produces adrenomedullin receptors, AM-1 and AM-2 receptors, respectively (3Kamitani S. Asakawa M. Shimekake Y. Kuwasako K. Nakahara K. Sakata T. FEBS Lett. 1999; 448: 111-114Crossref PubMed Scopus (112) Google Scholar, 4Buhlmann N. Leuthauser K. Muff R. Fischer J.A. Born W. Endocrinology. 1999; 140: 2883-2890Crossref PubMed Scopus (145) Google Scholar). AM and CGRP are multifunctional peptides with many overlapping functions, ranging from potent vasodilation to proliferation regulation to regulation of salt and water balance (5Hinson J.P. Kapas S. Smith D.M. Endocr. Rev. 2000; 21: 138-167Crossref PubMed Scopus (695) Google Scholar). Differential expression of RAMP isoforms has been hypothesized to play a regulatory role in both physiological and pathophysiological disease states. Moreover, the recent identification of RAMP interactions with additional members of the Class II GPCR family and RAMP expression in cell lines lacking CRLR have raised the possibility of novel functions for RAMPs in GPCR regulation (6Christopoulos A. Christopoulos G. Morfis M. Udawela M. Laburthe M. Couvineau A. Kuwasako K. Tilakaratne N. Sexton P.M. J. Biol. Chem. 2003; 278: 3293-3297Abstract Full Text Full Text PDF PubMed Scopus (255) Google Scholar). Upon activation, the CRLR-RAMP receptor complex causes cyclic AMP activation in most systems, irrespective of whether the ligand is AM or CGRP. In addition, the receptor complex undergoes desensitization and internalization (via clathrin-mediated endocytosis) in response to a prolonged agonist stimulation (7Hilairet 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 (128) Google Scholar). Once internalized, the receptor complex either undergoes degradation or recycling, depending on the cell type. In HEK293 cells the CRLR-RAMP complex has been shown to be targeted to the lysosomes for degradation, while in rat mesangial cells, the CRLR-RAMP receptor complex is sorted for dephosphorylation and resensitization (and presumably recycling) as a fully functional receptor (2Kuwasako 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 (145) Google Scholar, 8Parameswaran N. Aiyar N. Wu H. Brooks D.P. Nambi P. Spielman W.S. Eur. J. Pharmacol. 2000; 407: 205-210Crossref PubMed Scopus (16) Google Scholar). The mechanism that regulates the pathway to which the receptor complex is targeted after agonist-induced internalization remains unknown. Factors influencing the sorting of receptors in the early endosomes are largely unknown, but some of the critical players are beginning to be identified for the GPCRs. It has been shown in other GPCR systems that interactions with PSD-95/Discs-large/ZO-1 homology (PDZ) domain proteins are responsible for altering the receptor-targeting after internalization (9Xia Z. Gray J.A. Compton-Toth B.A. Roth B.L. J. Biol. Chem. 2003; 278: 21901-21908Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar, 10Li J.G. Chen C. Liu-Chen L.Y. J. Biol. Chem. 2002; 277: 27545-27552Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar, 11Cong M. Perry S.J. Hu L.A. Hanson P.I. Claing A. Lefkowitz R.J. J. Biol. Chem. 2001; 276: 45145-45152Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar). The life cycle of the β2-adrenergic receptor (β2-AR) was reported to be altered in the presence of a protein termed N-ethylmaleimide-sensitive factor (NSF) (11Cong M. Perry S.J. Hu L.A. Hanson P.I. Claing A. Lefkowitz R.J. J. Biol. Chem. 2001; 276: 45145-45152Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar). It has been shown that the β2-AR interacts with NSF via a PDZ type I motif (-DSLL) at its extreme C terminus. In addition, binding of NSF to the Glu2 subunits of the α-amino-3-hydroxy-5-methylisoxazolepropionate (AMPA) receptor was also demonstrated to be crucial for the recycling of the AMPA receptor (12Song I. Kamboj S. Xia J. Dong H. Liao D. Huganir R.L. Neuron. 1998; 21: 393-400Abstract Full Text Full Text PDF PubMed Scopus (353) Google Scholar, 13Nishimune A. Isaac J.T. Molnar E. Noel J. Nash S.R. Tagaya M. Collingridge G.L. Nakanishi S. Henley J.M. Neuron. 1998; 21: 87-97Abstract Full Text Full Text PDF PubMed Scopus (468) Google Scholar). NSF is a hexameric ATPase that plays a chaperoning role for soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) in the majority of membrane fusion events in a cell, but when targeting membrane receptors for recycling, NSF acts independently of the SNARE complex to promote rapid resensitization of the receptors at the plasma membrane (14Lin R.C. Scheller R.H. Annu. Rev. Cell Dev. Biol. 2000; 16: 19-49Crossref PubMed Scopus (422) Google Scholar, 15Neuwald A.F. Struct. Fold. Des. 1999; 7: R19-R23Abstract Full Text Full Text PDF Scopus (21) Google Scholar, 16Brunger A.T. Curr. Opin. Neurobiol. 2000; 10: 293-302Crossref PubMed Scopus (82) Google Scholar). Similar to the C terminus of β2-AR, human RAMP3 C terminus has a type-I PDZ motif (-DTLL motif). CRLR, RAMP1, or RAMP2 do not, however, contain any PDZ motifs. We hypothesized that RAMP3, via its interaction with NSF, regulates the trafficking of the CRLR-RAMP3 complex. We show here that while CRLR-RAMP1 and CRLR-RAMP2 complexes do not interact with NSF, CRLR-RAMP3 complex interacts with NSF via the PDZ motif of RAMP3. Moreover, we demonstrate that overexpression of NSF in HEK293 cells alters the life cycle of CRLR-RAMP3 complex from a degradative to recycling pathway via interactions of the PDZ motif of RAMP3 and NSF. These findings demonstrate that RAMP3, in addition to determining the receptor phenotype and allowing receptor membrane expression, is also significantly involved with the regulation of the turnover of the CRLR-RAMP complex. Materials—Adrenomedullin was purchased from Bachem Bioscience, Inc. (King of Prussia, PA). 125I-Labeled adrenomedullin was purchased from Amersham Biosciences. N-Ethylmaleimide was purchased from Sigma. Cell culture media, fetal bovine serum, penicillin/streptomycin, and trypsin-EDTA were purchased from Invitrogen. RAMP3 antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA), and NSF antibody was from Calbiochem. Anti-mouse Cy3 and anti-rabbit Cy5 secondary antibodies were from Jackson Immunoresearch Laboratories (West Grove, PA). All other reagents were of highest quality available. Cell Culture and Transfection Protocols—Rat mesangial cells were obtained from ATCC and are maintained in RMPI 1640 media containing 15% FBS and 1% penicillin-streptomycin. HEK293T cells (obtained from ATCC) are maintained in DMEM low glucose media containing 10% FBS, 1% penicillin-streptomycin. Rat-2 fibroblast cells (obtained from ATCC) are maintained in DMEM high glucose media containing 10% FBS, 1% penicillin-streptomycin. Transfection of HEK293T and Rat-2 fibroblast cells was performed using Lipofectamine Plus protocol (Invitrogen). Cells were transfected with the DNA and Lipofectamine Plus as per manufacturer's protocol. Cells were collected for assays after 48 h of transfection. RAMP Cloning and Expression—Full-length cDNA of human RAMP1, -2, and -3 and bovine CRLR were described before (17Aiyar N. Disa J. Ao Z. Xu D. Surya A. Pillarisetti K. Parameswaran N. Gupta S.K. Douglas S.A. Nambi P. Biochem. Pharmacol. 2002; 63: 1949-1959Crossref PubMed Scopus (14) Google Scholar, 18Nowak W. Parameswaran N. Hall C.S. Aiyar N. Sparks H.V. Spielman W.S. Am. J. Physiol. 2002; 282: C1322-C1331Crossref PubMed Scopus (15) Google Scholar). CRLR, cloned into N1-EGFP and also in pcDNA3.1 expression vectors, was used for transfection in HEK293T cells. Desensitization and Resensitization Assays—48 h post-tranfection cells were pretreated with or without 10 nm ADM in DMEM containing 0.2% BSA for indicated time periods (up to 4 h). After agonist exposure, cells were washed three times with Dulbecco's phosphate-buffered saline (dPBS, Invitrogen) containing 0.2% BSA and either frozen for membrane preparation for adenylate cyclase assays or used immediately for intact-cell radioligand binding. For receptor resensitization assays, after agonist exposure, cells were washed and incubated for indicated time periods in DMEM containing 0.2% BSA and 5 μg/ml cycloheximide to allow receptor recovery. Receptor Binding—Competition radioligand binding assays were performed as described by Aiyar et al. (19Aiyar N. Nambi P. Whitman M. Stassen F.L. Crooke S.T. Mol. Pharmacol. 1987; 31: 180-184PubMed Google Scholar) and as established in our laboratory. HEK293T cells were transfected and ∼200,000 cells/well seeded in poly-d-lysine precoated 24-well plates (BD Biosciences). 48 h post-tranfection cells were treated for desensitization or resensitization assays as described above. After agonist exposure, cells were washed three times with dPBS buffer containing 0.2% BSA then incubated with increasing concentrations (1 pm to 100 nm) of competing ligand and 175–250 pm125I-labeled rADM for 30 min at 37 °C. After incubation, plates were washed three times with ice-cold assay buffer and the reactions were terminated by the addition of 2 m NaOH. Cells were then harvested, and associated radioligand activity was counted on a γ-counter. All binding assays were performed in duplicate, with each experiment repeated at least three times. Nonspecific binding was determined in the presence of 100 nm of unlabeled rADM. Analysis of all binding data were performed by computer-assisted nonlinear least square fitting using GraphPad PRIZM (GraphPad Software, San Diego, CA). Adenylate Cyclase Assays—Cyclase activity was done as described before with slight modifications (18Nowak W. Parameswaran N. Hall C.S. Aiyar N. Sparks H.V. Spielman W.S. Am. J. Physiol. 2002; 282: C1322-C1331Crossref PubMed Scopus (15) Google Scholar). Cells were harvested from P100 or P60 plates and homogenized in Tris-HCl (10 mm), EDTA (10 mm) buffer. Membranes were prepared by homogenization and centrifugation in Tris-HCl (50 mm), MgCl (10 mm) buffer. Final concentration of 20 μg of protein/assay tube was obtained. Membranes were incubated for 15 min at 30 °C with appropriate concentrations of drugs and assay mix containing ATP regeneration system and [α-32P]ATP. After the reaction was stopped (with stop solution containing [3H]cAMP) contents of the assay tubes were passed through Dowex and subsequently through alumina columns to separate the degradation products of ATP, by washing the Dowex with water and alumina with imidazole. Elution profile was done to determine the amount of water and imidazole needed to wash and elute the products. Product eluted from alumina column was counted for the presence of [3H]cAMP and [α-32P]cAMP in a β-counter. Each experiment was done in triplicate and repeated at least three times. Data are expressed as percent maximal response, % forskolin. cAMP Accumulation Assays—Rat mesangial cells were seeded on a 24-well plate until reaching 80–90% confluence, then incubated in serum-free medium overnight before experiment. Resensitization experiments were carried out as described under "Experimental Procedures," with cells pretreated with 10 nm rAM and subsequently challenged with 100 nm rAM in the presence of 200 μm 3-isobutyl-1-methylxanthine. Determination of the cAMP level was measured using the Biotrak cAMP enzyme immunoassay system (Amersham Biosciences) according to the manufacturer's instructions. cAMP levels in rat mesangial cells were calculated using a standard curve ranging from 10 to 104 fmol of cAMP. Each experiment was done in duplicate and repeated at least three times. Data are expressed as percent maximal response, % forskolin. RNA Interference Analysis—Gene-specific d-siRNA for lacZ (control) and RAMP3 were generated and purified using BLOCK-iT Dicer RNAi kit from Invitrogen. Rat mesangial cells (RMCs) were transfected with d-siRNAs using Lipofectamine 2000 as per manufacturer's instructions (Invitrogen). 48 h after transfection cells were frozen for mRNA analysis or used for cAMP accumulation assays or immunofluorescence microscopy. Quantitative PCR Analysis—Total RNA was isolated from RMCs using TRIzol reagent (Invitrogen). After sodium acetate-ethanol precipitation and several ethanol washes, RNA was used as a template in a quantitative PCR amplification procedure. Quantitative PCR analysis was carried out with the LUX (light upon extension) fluorogenic primer method following the protocol in the manufacturer's manual (Invitrogen), as described by Nazarenko et al. (20Nazarenko I. Pires R. Lowe B. Obaidy M. Rashtchian A. Nucleic Acids Res. 2002; 30: 2089-2195Crossref PubMed Google Scholar). Mutagenesis Procedure—Site-directed mutagenesis was performed using a PCR-based strategy that employs the pfu Turbo polymerase (Stratagene, La Jolla, CA). A pair of complementary oligonucleotides containing the appropriate point mutations in the sequence of RAMP or a premature stop codon at position 145 or 147 codon of RAMP-3 for deletion mutants were synthesized (Michigan State University Macromolecular Structure Facility). The PCR for the mutation was as follows: 94 °C for 2 min; 30 cycles of 94 °C for 30 s, 50 °C for 30 s, 68 °C for 8 min; final cycle of 68 °C for 8 min. PCR product was digested for 4 h with DpnI enzyme (Invitrogen) and transformed in to DH5α cells. Mutations were confirmed by automated sequencing (Michigan State University Genomic Technology Support Facility). Immunofluorescence Microscopy—HEK293 cells were transfected as described above and seeded at 24 h post-transfection onto collagen type I-coated coverslips. Resensitization assays were performed as described and reactions were stopped by fixing cells in 4% paraformaldehyde for 30 min at room temperature. Samples were permeablized with 0.1% v/v Triton X-100 in PBS and blocked overnight in 0.1% v/v Triton X-100 in PBS + 10% goat serum. Samples were incubated in primary antibody in blocking buffer for 2 h at room temperature (NSF at 1:250 and RAMP3 at 1:200). Appropriate secondary antibodies were applied for 1 h at room temperature (goat anti-mouse Cy3 at 1:500 and goat anti-rabbit Cy5 at 1:400). Coverslips were mounted in Shandon Permafluor mounting medium and slides stored at 2–8 °C until analysis. Cells were visualized on a Zeiss 210 laser confocal microscope at a zoom of 2. Images presented are representative single optical sections of a z-series taken from at least 20 fields per experiment and at least three individual experiments. Fusion Protein Overlays and Western Blotting—10 μg of GST fusion proteins were resolved on a 10% SDS-PAGE gel and transferred to nitrocellulose filters. Filters were blocked with 5% w/v fat-free milk powder in Tris-buffered saline with Tween 20 (TTBS: 20 mm Tris, pH 7.4, 500 mm NaCl, 0.1% v/v Tween 20) and incubated overnight at 4 °C in lysates of HEK293 cells with or without overexpression NSF. Blots were then washed three times with TTBS buffer and incubated with anti-NSF monoclonal antibody for 2 h at room temperature. After three washes with TTBS, filters were incubated for 1 h with horseradish peroxidase-conjugated goat anti-mouse secondary antibody (Invitrogen), washed again with TTBS, soaked in SuperSignal West Pico chemiluminescent substrate (Pierce) and exposed to x-ray film. Same protocol, with the exception of the overnight incubation with cell lysate, was followed for immunoblot analysis of RAMP3. Statistics—Data are presented as mean ± S.E. Single group comparisons exercised a paired Student's t test method. Statistical significance was set at p < 0.05. Role of NSF in Resensitization of the CRLR-RAMP Complex—Our laboratory has previously published that RMCs endogenously express the AM1R (CRLR + RAMP2) and the AM2R (CRLR + RAMP3) (8Parameswaran N. Aiyar N. Wu H. Brooks D.P. Nambi P. Spielman W.S. Eur. J. Pharmacol. 2000; 407: 205-210Crossref PubMed Scopus (16) Google Scholar). These data were repeated, and NSF expression was confirmed in the RMCs with reverse transcription-PCR and immunocytochemistry (data not shown). Our laboratory has also reported that pretreatment of RMCs with AM leads to an agonist-stimulated desensitization and internalization of the CRLR-RAMP complex. Phosphatase-dependent resensitization of AM responsiveness was also demonstrated after agonist-stimulated desensitization (8Parameswaran N. Aiyar N. Wu H. Brooks D.P. Nambi P. Spielman W.S. Eur. J. Pharmacol. 2000; 407: 205-210Crossref PubMed Scopus (16) Google Scholar). Measuring cAMP accumulation we repeated these results in this study (Fig. 1). As a preliminary test to determine whether NSF is involved in the resensitization of AM responsiveness, we used a pharmacological inhibitor of NSF, N-ethylmaleimide (NEM). In RMCs treated for 45 s with 50 μm NEM during the resensitization experiment, resensitization was blocked, as measured by cAMP accumulation (Fig. 1). NEM, however, did not affect basal cAMP accumulation or the desensitization response when compared with untreated cells, an important finding given the ability of NEM to interfere with Gα subunits (Fig. 1). These results indicate that NSF plays a role in the sorting of the CRLR-RAMP complex following agonist-induced internalization in this endogenous CRLR-RAMP system where the receptor complex is recycled. To fully evaluate the molecular mechanisms of this observation, we used HEK293 cells to examine the interaction of the CRLR-RAMP complex with NSF and the impact of this interaction on receptor trafficking. In contrast to RMCs, HEK293 cells express very low endogenous levels of RAMPs. Kuwasako et al. (2Kuwasako 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 (145) Google Scholar) have demonstrated that in HEK293 cells overexpressing the CRLR-RAMP complex, agonist-induced internalization leads to receptor trafficking to a degradation pathway. In this study the internalized CRLR/RAMP complexes were colocalized with LAMP-1, a lysosomal marker, to show the targeting of the receptor for the degradation pathway. Utilizing adenylate cyclase activity assays, whole-cell ligand binding, and immunofluorescence microscopy we confirmed these findings (Fig. 2, A and B). Pretreatment of HEK293 cells transfected with CRLR and RAMP3 with 10 nm AM for 1 h resulted in desensitization of the adenylate cyclase response from 50% (of forskolin stimulation) in untreated cells to 28% in AM-treated cells (Fig. 2A left axis). Even after the removal of agonist and incubation with buffer alone for indicated times through 4 h, the adenylate cyclase response remained desensitized (Fig. 2A, left axis), indicating a lack of resensitization. Consistent findings were obtained with whole-cell binding and immunofluorescence microscopy experiments (Fig. 2, A, right axis, and B). To determine whether NSF overexpression could alter the receptor trafficking in this cell system, NSF was co-transfected with CRLR and RAMP3, and resensitization and recycling assays were performed. Resensitization and recycling were monitored by adenylate cyclase activity assays and whole-cell competition binding, respectively. In addition, visualization of the trafficking of the receptor complex was performed by immunofluorescence microscopy. In the absence of NSF, pretreatment with AM for 1 h resulted in desensitization of the adenylate cyclase response and internalization of the receptor complex. Upon removal of agonist and incubation with buffer alone for 4 h, the adenylate cyclase response remained desensitized and the receptor complex remained internalized, indicating a lack of resensitization (Fig. 3, A and B). In contrast, when NSF was co-transfected in the cells, although the desensitization response (i.e. response after 1-h agonist treatment) was not altered, the cells now underwent time-dependent resensitization (i.e. response after 1-, 2-, or 4-h agonist removal) in response to AM (Fig. 3B). Consistent findings were obtained with whole-cell binding and immunofluorescence microscopy experiments (Figs. 3A and 4). Time course experiments indicated the time course for complete resensitization and recycling of the CRLR-RAMP3 receptor complex to be 4 h in HEK293 cells, as measured by adenylate cyclase, whole-cell binding, and immunofluorescence microscopy experiments (Figs. 3 and 4). All subsequent experiments in HEK293 cells use the 4 h time point to determine receptor complex recycling and resensitization. These results indicate that the presence of NSF alters the intracellular sorting of the CRLR/RAMP3 receptor complex after AM-stimulated endocytosis.Fig. 4Localization of CRLR, RAMP3, and NSF in HEK293 cells during a recycling experiment.A, after 1-h ADM pretreatment, CRLR and RAMP3 are internalized and show co-localization with NSF intracellularly. After a 4 h recovery time post-ADM pretreatment, CRLR and RAMP3 show distribution at the plasma membrane of the cell, demonstrating recycling of the receptor complex. HEK293 cells transfected with CRLR-GFP, RAMP3, and NSF were pretreated with 10 nm ADM for 1 h. After pretreatment with ADM, cells were washed and incubated in serum-free medium with 5 μg/ml cycloheximide to allow receptor recycling for indicated times. Note: "1 hr Pretreatment" indicates time just after ADM pretreatment and wash steps, with no recovery time. Cells were fixed and components were visualized using anti-RAMP3 antibody (1:200) and anti-NSF antibody (1:250) with Cy5 anti-rabbit secondary antibody (1:400, in blue) and Cy3 anti-mouse secondary antibody (1:500, in red), respectively; CRLR-GFP is detected with an EGFP tag and shown in green; overlays of staining patterns are shown in the far right panels. Images shown are representative of at least 20 fields imaged per experiment from at least three experiments. Bar scales on all images represent 100 μm.View Large Image Figure ViewerDownload Hi-res image Download (PPT) RAMP Isoform-specific Regulation of CRLR-RAMP Receptor Complex Trafficking—To determine whether this effect of NSF was specific for RAMP3, the additional RAMPs (RAMP1 or RAMP2) were tested for their ability to act with NSF to alter the receptor complex life cycle. Interestingly, in contrast to RAMP3, presence of NSF did not alter the resensitization response or recycling pattern of the CRLR/RAMP1 or RAMP2 receptor complexes. Both the activity and receptor number remained at desensitized levels in cells transfected with CRLR+RAMP1 or CRLR+RAMP2 (along with NSF) (Fig. 5, A–D). Both CRLR/RAMP1 and CRLR/RAMP2 complexes showed no difference in receptor expression levels at the plasma membrane (as measured with whole-cell binding) in untreated cells, as compared with CRLR/RAMP3 complex. These results indicate that RAMP3 must contain a