Helix 8 Leu in the CB1 Cannabinoid Receptor Contributes to Selective Signal Transduction Mechanisms
2007; Elsevier BV; Volume: 282; Issue: 34 Linguagem: Inglês
10.1074/jbc.m703388200
ISSN1083-351X
AutoresSharon Anavi‐Goffer, Daniel Fleischer, Dow P. Hurst, Diane L. Lynch, Judy Barnett‐Norris, Shanping Shi, Deborah L. Lewis, Somnath Mukhopadhyay, Allyn C. Howlett, Patricia H. Reggio, Mary E. Abood,
Tópico(s)Pancreatic function and diabetes
ResumoThe intracellular C-terminal helix 8 (H8) of the CB1 cannabinoid receptor deviates from the highly conserved NPXXY(X)5,6F G-protein-coupled receptor motif, possessing a Leu instead of a Phe. We compared the signal transduction capabilities of CB1 with those of an L7.60F mutation and an L7.60I mutation that mimics the CB2 sequence. The two mutant receptors differed from wild type (WT) in their ability to regulate G-proteins in the [35S]guanosine 5′-3-O-(thio)triphosphate binding assay. The L7.60F receptor exhibited attenuated stimulation by agonists WIN-55,212-2 and CP-55,940 but not HU-210, whereas the L7.60I receptor exhibited impaired stimulation by all agonists tested as well as by the inverse agonist rimonabant. The mutants internalized more rapidly than WT receptors but could equally sequester G-proteins from the somatostatin receptor. Both the time course and maximal N-type Ca2+ current inhibition by WIN-55,212-2 were reduced in the mutants. Reconstitution experiments with pertussis toxin-insensitive G-proteins revealed loss of coupling to Gαi3 but not Gα0A in the L7.60I mutant, whereas the reduction in the time course for the L7.60F mutant was governed by Gαi3. Furthermore, Gαi3 but not Gα0A enhanced basal facilitation ratio, suggesting that Gαi3 is responsible for CB1 tonic activity. Co-immunoprecipitation studies revealed that both mutant receptors were associated with Gαi1 or Gαi2 but not with Gαi3. Molecular dynamics simulations of WT CB1 receptor and each mutant in a 1-palmitoyl-2-oleoylphosphatidylcholine bilayer suggested that the packing of H8 is different in each. The hydrogen bonding patterns along the helix backbones of each H8 also are different, as are the geometries of the elbow region of H8 (R7.56(400)-K7.58(402)). This study demonstrates that the evolutionary modification to NPXXY(X)5,6L contributes to maximal activity of the CB1 receptor and provides a molecular basis for the differential coupling observed with chemically different agonists. The intracellular C-terminal helix 8 (H8) of the CB1 cannabinoid receptor deviates from the highly conserved NPXXY(X)5,6F G-protein-coupled receptor motif, possessing a Leu instead of a Phe. We compared the signal transduction capabilities of CB1 with those of an L7.60F mutation and an L7.60I mutation that mimics the CB2 sequence. The two mutant receptors differed from wild type (WT) in their ability to regulate G-proteins in the [35S]guanosine 5′-3-O-(thio)triphosphate binding assay. The L7.60F receptor exhibited attenuated stimulation by agonists WIN-55,212-2 and CP-55,940 but not HU-210, whereas the L7.60I receptor exhibited impaired stimulation by all agonists tested as well as by the inverse agonist rimonabant. The mutants internalized more rapidly than WT receptors but could equally sequester G-proteins from the somatostatin receptor. Both the time course and maximal N-type Ca2+ current inhibition by WIN-55,212-2 were reduced in the mutants. Reconstitution experiments with pertussis toxin-insensitive G-proteins revealed loss of coupling to Gαi3 but not Gα0A in the L7.60I mutant, whereas the reduction in the time course for the L7.60F mutant was governed by Gαi3. Furthermore, Gαi3 but not Gα0A enhanced basal facilitation ratio, suggesting that Gαi3 is responsible for CB1 tonic activity. Co-immunoprecipitation studies revealed that both mutant receptors were associated with Gαi1 or Gαi2 but not with Gαi3. Molecular dynamics simulations of WT CB1 receptor and each mutant in a 1-palmitoyl-2-oleoylphosphatidylcholine bilayer suggested that the packing of H8 is different in each. The hydrogen bonding patterns along the helix backbones of each H8 also are different, as are the geometries of the elbow region of H8 (R7.56(400)-K7.58(402)). This study demonstrates that the evolutionary modification to NPXXY(X)5,6L contributes to maximal activity of the CB1 receptor and provides a molecular basis for the differential coupling observed with chemically different agonists. The CB1 cannabinoid receptor is a member of the class A G-protein-coupled receptor (GPCR) 3The abbreviations used are: GPCRG-protein-coupled receptorCBcannabinoidWTwild typeTMHtransmembrane helixH8helix 8ICintracellular loopHEK293human embryonic kidney 293SCGsuperior cervical ganglionGTPγSguanosine 5′-3-O-(thio)triphosphatePOPC1-palmitoyl-2-oleoylphosphatidylcholinePTXpertussis toxinCHAPS3-[(3-chlamidopropyl)dimethylammonio]-1-propanesulfonic acidCAPS3-(cyclohexylamino)propanesulfonic acidVMDvisual molecular dynamicshhuman 3The abbreviations used are: GPCRG-protein-coupled receptorCBcannabinoidWTwild typeTMHtransmembrane helixH8helix 8ICintracellular loopHEK293human embryonic kidney 293SCGsuperior cervical ganglionGTPγSguanosine 5′-3-O-(thio)triphosphatePOPC1-palmitoyl-2-oleoylphosphatidylcholinePTXpertussis toxinCHAPS3-[(3-chlamidopropyl)dimethylammonio]-1-propanesulfonic acidCAPS3-(cyclohexylamino)propanesulfonic acidVMDvisual molecular dynamicshhuman superfamily (see Fig. 1). The CB1 receptor is activated by endocannabinoid lipid mediators (anandamide, 2-arachidonoylglycerol), as well as cannabinoid (HU-210, CP-55940) and aminoalkylindole (WIN-55,212-2) agonists, leading to diverse signaling responses (1Howlett A.C. Barth F. Bonner T.I. Cabral G. Casellas P. Devane W.A. Felder C.C. Herkenham M. Mackie K. Martin B.R. Mechoulam R. Pertwee R.G. Pharmacol. Rev. 2002; 54: 161-202Crossref PubMed Scopus (2302) Google Scholar). CB1 receptor agonists can selectively regulate G-protein coupling (2Houston D.B. Howlett A.C. Cell. Signal. 1998; 10: 667-674Crossref PubMed Scopus (53) Google Scholar, 3Glass M. Northup J. Mol. Pharmacol. 1999; 56: 1362-1369Crossref PubMed Scopus (245) Google Scholar, 4Mukhopadhyay S. McIntosh H. Houston D. Howlett A. Mol. Pharmacol. 2000; 57: 162-170PubMed Google Scholar, 5Mukhopadhyay S. Howlett A.C. Eur. J. Biochem. 2001; 268: 499-505Crossref PubMed Scopus (75) Google Scholar). Competitive antagonists, such as the arylpyrazole rimonabant (also known as SR141716), can inhibit CB1 constitutive activity and thereby behave as inverse agonists (6Pan X. Ikeda S. Lewis D. Mol. Pharmacol. 1998; 54: 1064-1072Crossref PubMed Scopus (168) Google Scholar, 7Bouaboula M. Perrachon S. Milligan L. Canat X. Rinaldi-Carmona M. Portier M. Barth F. Calandra B. Pecceu F. Lupker J. Maffrand J.P. Le Fur G. Casellas P. J. Biol. Chem. 1997; 272: 22330-22339Abstract Full Text Full Text PDF PubMed Scopus (422) Google Scholar, 8Sim-Selley L.J. Brunk L.K. Selley D.E. Eur. J. Pharmacol. 2001; 414: 135-143Crossref PubMed Scopus (84) Google Scholar). However, the protein conformation signals that underlie the ligand-directed recognition of the CB1 receptor for different G-protein subtypes and their activation are not understood. G-protein-coupled receptor cannabinoid wild type transmembrane helix helix 8 intracellular loop human embryonic kidney 293 superior cervical ganglion guanosine 5′-3-O-(thio)triphosphate 1-palmitoyl-2-oleoylphosphatidylcholine pertussis toxin 3-[(3-chlamidopropyl)dimethylammonio]-1-propanesulfonic acid 3-(cyclohexylamino)propanesulfonic acid visual molecular dynamics human G-protein-coupled receptor cannabinoid wild type transmembrane helix helix 8 intracellular loop human embryonic kidney 293 superior cervical ganglion guanosine 5′-3-O-(thio)triphosphate 1-palmitoyl-2-oleoylphosphatidylcholine pertussis toxin 3-[(3-chlamidopropyl)dimethylammonio]-1-propanesulfonic acid 3-(cyclohexylamino)propanesulfonic acid visual molecular dynamics human Crystal structures of bovine rhodopsin (9Palczewski K. Kumasaka T. Hori T. Behnke C.A. Motoshima H. Fox B.A. Le Trong I. Teller D.C. Okada T. Stenkamp R.E. Yamamoto M. Miyano M. Science. 2000; 289: 739-745Crossref PubMed Scopus (5023) Google Scholar, 10Li J. Edwards P.C. Burghammer M. Villa C. Schertler G.F. J. Mol. Biol. 2004; 343: 1409-1438Crossref PubMed Scopus (673) Google Scholar), a prototype member of class A GPCRs, reveal an intracellular α-helical extension of transmembrane helix (TMH) 7 (Lys311 to Cys323). This domain, referred to as helix 8 (H8), is positioned along the intracellular surface of TMH1 (9Palczewski K. Kumasaka T. Hori T. Behnke C.A. Motoshima H. Fox B.A. Le Trong I. Teller D.C. Okada T. Stenkamp R.E. Yamamoto M. Miyano M. Science. 2000; 289: 739-745Crossref PubMed Scopus (5023) Google Scholar, 10Li J. Edwards P.C. Burghammer M. Villa C. Schertler G.F. J. Mol. Biol. 2004; 343: 1409-1438Crossref PubMed Scopus (673) Google Scholar). Photoactivation of rhodopsin has been associated with movement of H8 as detected in cysteine cross-linking studies and in nitroxide spin label studies (11Altenbach C. Klein-Seetharaman J. Cai K. Khorana H.G. Hubbell W.L. Biochemistry. 2001; 40: 15493-15500Crossref PubMed Scopus (104) Google Scholar). The highly conserved NPXXY(X)5,6F motif of rhodopsin has been proposed to provide structural constraints via the aromatic interaction of Y7.53(306) in the NPXXY motif of TMH7 to F7.60(313) in H8, which rearrange in response to photo-isomerization (12Fritze O. Filipek S. Kuksa V. Palczewski K. Hofmann K.P. Ernst O.P. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 2290-2295Crossref PubMed Scopus (294) Google Scholar). This same interaction has been shown to be important for the switching of the 5HT-2C receptor among multiple active and inactive conformations (13Prioleau C. Visiers I. Ebersole B.J. Weinstein H. Sealfon S.C. J. Biol. Chem. 2002; 277: 36577-36584Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). Taken together, these studies suggest that the H8 domain plays a role in docking specific G-protein subunits and in the transition between conformational states. The CB1 receptor possesses a defined intracellular helical segment (H8 D7.59(403) to P7.69(413)), as predicted by Fourier transform analysis of the primary sequence of the CB1 receptor (14Bramblett R.D. Panu A.M. Ballesteros J.A. Reggio P.H. Life Sci. 1995; 56: 1971-1982Crossref PubMed Scopus (100) Google Scholar). Significant helicity was demonstrated when the CB1 401–417 peptide was present in an anionic micelle environment (SDS or phosphatidic acid) but not in aqueous media (15Mukhopadhyay S. Cowsik S. Lynn A. Welsh W. Howlett A. Biochemistry. 1999; 38: 3447-3455Crossref PubMed Scopus (39) Google Scholar). Recent NMR and circular dichroism studies also revealed significant α-helical structure of synthetic peptides in dodecyl phosphocholine micelles (16Choi G. Guo J. Makriyannis A. Biochim. Biophys. Acta. 2005; 1668: 1-9Crossref PubMed Scopus (39) Google Scholar, 17Xie X.Q. Chen J.Z. J. Biol. Chem. 2005; 280: 3605-3612Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar) or SDS (18Grace C.R. Cowsik S.M. Shim J.Y. Welsh W.J. Howlett A.C. J. Struct. Biol. 2007; PubMed Google Scholar). However, the CB1 receptor differs from the NPXXY(X)5,6F motif, as amino acid residue 7.60(404) is a Leu in CB1. We explored the ramifications of this deviation from the highly conserved NPXXY(X)5,6F motif, by comparing the signal transduction capabilities of the CB1 wild-type with those of an L7.60F mutation which mimics the rhodopsin motif, and an L7.60I mutation which mimics the homologous CB2 receptor sequence. We report here that the CB1 receptor signal transduction profiles are considerably altered by substitution of the amino acid residue at position 7.60 of H8. Activation of G-proteins by agonists was attenuated in both mutants stably expressed in HEK293 cells. Only the L7.60I mutant showed a loss of response to the inverse agonist rimonabant. Furthermore, L7.60 plays a crucial role in modulating the rate of agonist-induced receptor internalization. Co-immunoprecipitation studies reveal that the CB1 receptor association with Gαi3 was abrogated in both mutant receptors, whereas the association with Gαi1 and Gαi2 remained intact. In superior cervical ganglion (SCG) neurons, N-type Ca2+ current inhibition by WIN-55,212-2 and the time to reach 90% of this inhibition was significantly reduced by both mutants. In addition, reconstitution experiments with pertussis toxin-insensitive G-proteins indicate that Gαi3 is responsible for CB1 tonic activity. Multiple nanosecond molecular dynamics simulations of WT CB1, and the L7.60F and L7.60I mutants, in a fully hydrated POPC phospholipid bilayer environment, suggest that the packing of H8 is different in each. Hence, the differential coupling to G-proteins and the alterations in the rate of internalization seen with these mutants may result from these variations in the H8 structure. Materials—[3H]CP-55,940 was purchased from PerkinElmer Life Sciences. WIN-55,212-2 was procured from Tocris. [3H]Rimonabant, rimonabant, and CP-55,940 were obtained from the National Institute on Drug Abuse. HU-210 (11-hydroxy-Δ8-THC dimethylheptyl) was a gift from Prof. Mechoulam (Hebrew University, Jerusalem, Israel). d-Trp8 somatosatin-14 was from Bachem. Anti-Gαi antibody was from Biomol. GDP and protease inhibitor mixture (4-(2-aminoethyl)benzenesulfonyl fluoride) were purchased from Sigma; GTPγS was from Roche Applied Science, and [35S]GTPγS and ECL reagents were from Amersham Biosciences. All G-protein subunits were obtained from UMR cDNA Resource Center (Rolla, MO). Pertussis toxin was from List Biological Laboratories, Campbell, CA. For radioligand binding assays, all the drugs were dissolved in ethanol. For [35S]GTPγS assays, drugs were dissolved in Me2SO. Mutagenesis—Mutations were introduced into the hCB1 receptor that had been subcloned into pcDNA3 vector (Invitrogen) with a QuikChange site-directed mutagenesis kit (Stratagene) as described previously (19McAllister S.D. Rizvi G. Anavi-Goffer S. Hurst D.P. Barnett-Norris J. Lynch D.L. Reggio P.H. Abood M.E. J. Med. Chem. 2003; 46: 5139-5152Crossref PubMed Scopus (178) Google Scholar). The hCB1 L7.60I mutation was made with the mutagenic primer (forward) TCGCTGAGGAGTAAGGACATCCGACACGCTTTCCGGAGC. The hCB1 L7.60F mutation was made with the mutagenic primer (forward) TCGCTGAGGAGTAAGGACTTCCGACACGCTTTCCGGAGC. The mutant cDNAs were sequenced to confirm the presence of the desired substitution. Cell Transfection—HEK293 cells were grown and stably transfected with expression plasmids containing WT or mutant hCB1 receptor sequences as described previously (19McAllister S.D. Rizvi G. Anavi-Goffer S. Hurst D.P. Barnett-Norris J. Lynch D.L. Reggio P.H. Abood M.E. J. Med. Chem. 2003; 46: 5139-5152Crossref PubMed Scopus (178) Google Scholar). Immunochemistry—Selected colonies were expanded and tested for receptor expression by immunochemistry as described previously (19McAllister S.D. Rizvi G. Anavi-Goffer S. Hurst D.P. Barnett-Norris J. Lynch D.L. Reggio P.H. Abood M.E. J. Med. Chem. 2003; 46: 5139-5152Crossref PubMed Scopus (178) Google Scholar). Immunofluorescence was visualized with a laser-scanning confocal fluorescence microscope (Nikon). In control experiments, no labeling was observed with the secondary antibody alone or when the primary antibody was incubated with CB1 receptor peptide at 100 μg/ml. For internalization studies, cells were preincubated with 1 μm CP-55,940 for 30 min or 1 h at 37 °C and then labeled (19McAllister S.D. Rizvi G. Anavi-Goffer S. Hurst D.P. Barnett-Norris J. Lynch D.L. Reggio P.H. Abood M.E. J. Med. Chem. 2003; 46: 5139-5152Crossref PubMed Scopus (178) Google Scholar). Radioligand Binding—As described previously (19McAllister S.D. Rizvi G. Anavi-Goffer S. Hurst D.P. Barnett-Norris J. Lynch D.L. Reggio P.H. Abood M.E. J. Med. Chem. 2003; 46: 5139-5152Crossref PubMed Scopus (178) Google Scholar), binding was initiated by the addition of 50 or 60 μg of membrane protein into tubes containing [3H]CP-55,940 (158 Ci/mmol) or [3H]rimonabant (16.9 Ci/mmol). Nonspecific binding was assessed by the addition of 1 μm unlabeled CP-55,940 or rimonabant to the tubes. [35S]GTPγS Binding Assay—Cells were harvested in phosphate-buffered saline containing 1 mm EDTA and centrifuged at 500 × g for 5 min, as described previously (20McAllister S.D. Hurst D.P. Barnett-Norris J. Lynch D. Reggio P.H. Abood M.E. J. Biol. Chem. 2004; 279: 48024-48037Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar). The cell pellet was homogenized and centrifuged at 50,000 × g, for 10 min at 4 °C. Binding was initiated by the addition of 10 μg (for agonist experiments) or 20 μg (for antagonist experiments) of membrane protein into glass tubes containing 0.1 nm [35S]GTPγS, 10 μm GDP in GTPγS binding buffer (100 mm NaCl, 3 mm MgCl2, 0.2 mm EGTA, 0.1% bovine serum albumin, pH 7.4). Nonspecific binding was assessed in the presence of 20 μm unlabeled GTPγS. Immunoprecipitation and Western Blot Analysis—HEK293 cells expressing WT, L7.60F, or L7.60I receptors were homogenized in a glass homogenizer in ice-cold buffer composed of 20 mm Na-HEPES (pH 8.0), 2 mm MgCl2, 1 mm EDTA, containing a protease inhibitor mixture (1.04 mm 4-(2-aminoethyl)benzenesulfonyl fluoride, 0.8 μm aprotinin, 20 μm leupeptin). After sedimentation at 1000 × g for 5 min at 4 °C to remove nuclear debris, the supernatant was collected and sedimented at 17,000 × g for 20 min at 4 °C. The pellet (P2 membrane fraction) was then solubilized using CHAPS hydrate detergent according to the method described by Houston and Howlett (2Houston D.B. Howlett A.C. Cell. Signal. 1998; 10: 667-674Crossref PubMed Scopus (53) Google Scholar). The immunoprecipitation of the CB1 receptor and associated proteins from the detergent-solubilized extract was performed as described previously (4Mukhopadhyay S. McIntosh H. Houston D. Howlett A. Mol. Pharmacol. 2000; 57: 162-170PubMed Google Scholar, 5Mukhopadhyay S. Howlett A.C. Eur. J. Biochem. 2001; 268: 499-505Crossref PubMed Scopus (75) Google Scholar). Immunoprecipitated samples were subjected to PAGE on 0.1% SDS, 10% polyacrylamide, 6 m urea gels. Electrophoretic transfer of proteins from the gel to polyvinylidene difluoride membranes was carried out in 10 mm CAPS buffer with 0.01% SDS (pH 11), for 12 h (0–4 °C) at 20 V using a Bio-Rad Trans-Blot cell. Blots were incubated with affinity-purified anti-CB1-1–14) combined with the indicated anti-Gαi antibody in blocking buffer for 3 h at room temperature (4Mukhopadhyay S. McIntosh H. Houston D. Howlett A. Mol. Pharmacol. 2000; 57: 162-170PubMed Google Scholar, 5Mukhopadhyay S. Howlett A.C. Eur. J. Biochem. 2001; 268: 499-505Crossref PubMed Scopus (75) Google Scholar). Neuron Preparation and Electrophysiological Recording of Ca2+ Currents—Superior cervical ganglion (SCG) neurons were isolated from adult male Wistar rats as described previously (21Nie J. Lewis D.L. J. Neurosci. 2001; 21: 8758-8764Crossref PubMed Google Scholar) with minor modifications. Briefly, isolated superior cervical ganglia were enzymatically digested and plated onto poly-l-lysine precoated 35-mm dishes in minimum essential medium with 10% fetal calf serum, 1% glutamine, and 1% penicillin/streptomycin. After 3–4 h at 37 °C in 5% CO2 plasmid cDNA (100 ng/μl) encoding hCB1, hCB1-L7.60I or hCB1-L7.60F was microinjected directly into the nucleus of single SCG neurons. The pEGFP-N1 plasmid (10 ng/μl) encoding the enhanced green fluorescent protein was used as a co-injection marker (21Nie J. Lewis D.L. J. Neurosci. 2001; 21: 8758-8764Crossref PubMed Google Scholar). For G-protein reconstitution experiments, pertussis toxin-insensitive GαoA(C351G) and Gαi3(C351G) subunits were each microinjected with β1 and γ2 at a ratio (mass) of 0.5:1.25:1.25. Neurons were treated overnight with 500 ng/ml pertussis toxin (PTX). Ca2+ currents were recorded from neurons at room temperature 16–20 h after injection using the whole-cell patch clamp technique (21Nie J. Lewis D.L. J. Neurosci. 2001; 21: 8758-8764Crossref PubMed Google Scholar). Ca2+ currents were elicited by voltage steps from −80 mV to +5 mV. A double-pulse protocol consisting of two 25-ms steps to +5 mV was used to elicit Ca2+ currents. The second voltage step to +5 mV was preceded by a 50-ms voltage step to +80 mV in order to remove the voltage-dependent, G-protein-mediated inhibition of the Ca2+ channels. Current amplitudes were measured isochronally as the average current between 10 and 11.8 ms after the voltage step to +5 mV. To isolate Ca2+ currents for whole-cell recording, cells were bathed in external and internal solutions as described previously (21Nie J. Lewis D.L. J. Neurosci. 2001; 21: 8758-8764Crossref PubMed Google Scholar). Neurons were superfused with external solution or with external solution containing d-Trp8 somatosatin-14 or WIN-55,212-2 using the SF-77B Perfusion Fast-Step device (Warner Instruments). Data and Statistical Analysis—Results are presented as means ± S.E. Data from radioligand binding and GTPγS assays were analyzed, and curves were generated through nonlinear regression analyses (or nonlinear analysis, when appropriate) with Prism software (Prism GraphPad, San Diego). Kd, Bmax, Emax, and EC50 values were calculated. Statistical analysis of variance (one-way analysis of variance) and the Bonferroni multiple comparison post hoc tests were performed for Bmax and Emax with GraphPad Prism. Two-tailed Student's t tests were performed on the log Kd values, log EC50 values ±95% confidence limits and for analyses of Ca2+ currents. p values <0.05 were defined as statistically significant. Amino Acid Numbering System—The amino acid numbering scheme proposed by Ballesteros and co-workers (14Bramblett R.D. Panu A.M. Ballesteros J.A. Reggio P.H. Life Sci. 1995; 56: 1971-1982Crossref PubMed Scopus (100) Google Scholar) was used. Sequence numbers used are human CB1 sequence numbers unless otherwise noted (14Bramblett R.D. Panu A.M. Ballesteros J.A. Reggio P.H. Life Sci. 1995; 56: 1971-1982Crossref PubMed Scopus (100) Google Scholar). Residues in the intracellular extension of TMH7 (H8) are numbered here as if they are part of TMH7 following the literature precedent set by Prioleau et al. (13Prioleau C. Visiers I. Ebersole B.J. Weinstein H. Sealfon S.C. J. Biol. Chem. 2002; 277: 36577-36584Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). WT CB1 Receptor Model Construction—Using the Loopy program within the protein structure modeling suite, Jackal 1.5 (J. Z. Xiang and B. Honig, Columbia University), extracellular (EC-1 His181–Ser185, EC-2 Cys257–Glu273, and EC-3 Gly369–Lys376) and intracellular loops (IC-1 Ser146–Arg150, IC-2 Pro221–Val228, and IC-3 Ala301–Pro332) as well as portions of the N and C termini were added to our previously built transmembrane helix (TMH) bundle model of the CB1 receptor (65Hurst D. Umijiego U. Lynch D. Seltzman H. Hyatt H. Roche M. McAllister S.D. Fleischer D. Kapur A. Abood M.E. Shi S. Jones J. Lewis D. Reggio P.H. J. Med. Chem. 2006; 49: 5969-5987Crossref PubMed Scopus (69) Google Scholar). The Modeler program was then used to refine loop structures (22Fiser A. Do R.K. Sali A. Protein Sci. 2000; 9: 1753-1773Crossref PubMed Scopus (1629) Google Scholar, 23Sali A. Blundell T.L. J. Mol. Biol. 1993; 234: 779-815Crossref PubMed Scopus (10512) Google Scholar). The chosen loop configurations for the final bundle model were those that produced a low value of the modeler objective function. Moreover, because of their close spatial proximity, the EC1 and EC3 loops were run concurrently. EC-2 Loop—One of the significant sequence divergences between rhodopsin and CB1 is in the second extracellular (EC-2) loop region. This loop in CB1 is shorter than in rhodopsin and is missing the conserved disulfide bridge between the cysteine in EC-2 and C3.25 in TMH 3 of rhodopsin. Instead, there is a Cys residue at the extracellular end of TMH4 in CB1 and a Cys near the middle of the EC-2 loop that experiments suggest may form a disulfide bridge (24Fay J.F. Dunham T.D. Farrens D.L. Biochemistry. 2005; 44: 8757-8769Crossref PubMed Scopus (48) Google Scholar). Consequently, the position of the EC-2 loop with respect to the binding site crevice in CB1 around TMHs 3–4–5 is likely to be quite different from that in rhodopsin. For this reason, the refined EC-2 loop (Cys257–Glu273) structure built by Modeler was removed, and the conformation of this loop was calculated using the Biased Scaled Collective Variable in Monte Carlo method (25Barnett-Norris J. Hurst D.P. Reggio P.H. 2003 Symposium on the Cannabinoids, Cornwall, Ontario, Canada, June 24–27, 2003. International Cannabinoid Research Society, Burlington, VT2003Google Scholar, 26Hassan S.A. Mehler E.L. Weinstein H. Gan H.H. Computational Methods for Macromolecules: Challenges and Applications. 24. Springer-Verlag Inc., New York2002: 197-231Google Scholar). The aqueous environment of the EC-2 loop was modeled during these calculations with a recently developed implicit solvent model that is based on a screened Coulomb potential formulation (the SCP-ISM) (27Hassan S.A. Guarnieri F. Mehler E.L. J. Phys. Chem. 2000; 104: 6478-6489Crossref Scopus (102) Google Scholar, 28Hassan S.A. Guarnieri F. Mehler E.L. J. Phys. Chem. 2000; 104: 6490-6498Crossref Scopus (49) Google Scholar). The EC-2 loop was modeled with an internal C4.66(257)-Cys264 disulfide bridge based upon mutation results from the Farrens laboratory (see Ref. 24Fay J.F. Dunham T.D. Farrens D.L. Biochemistry. 2005; 44: 8757-8769Crossref PubMed Scopus (48) Google Scholar), which show that these two cysteines are required for high level expression and receptor function. IC-3 Loop—The CB1 IC-3 loop is much longer than the corresponding sequence in rhodopsin. NMR experiments have been performed on a peptide fragment comprised of the CB1 sequence span from the intracellular end of TMH5 to the intracellular end of TMH6 in micelles (29Ulfers A.L. McMurry J.L. Miller A. Wang L. Kendall D.A. Mierke D.F. Protein Sci. 2002; 11: 2526-2531Crossref PubMed Scopus (34) Google Scholar). This study suggested that part of the IC-3 loop is α-helical. This region occurs after the intracellular end of TMH5 (K5.64(300)) and consists of a short α-helical segment from Ala301 to Arg307, followed by an elbow region (Arg307–Ile309) and an α-helical segment (Gln310–Ser316) up to a Ile–Ile–Ile (Ile317–Ile319) in IC-3. Based on these results, we replaced the initial Modeler-built IC-3 loop with this α-helix-elbow-α-helix region, and then the rest of IC-3 loop (Ile317–Pro332) was re-built and optimized using Modeler. N and C Termini—The N and C termini were added last to the model. The N terminus of CB1 is 111 residues in length. A shorter portion of this terminus from Asn95 to Asn112 (NIQC-GENFMDIECFMVLN) was added to the CB1 model using Modeler. A C-terminal fragment Ser414–Gly427 (SCEGTAQ-PLDNS-MG), which contains a putative palmitoylation site at Cys415 (24Fay J.F. Dunham T.D. Farrens D.L. Biochemistry. 2005; 44: 8757-8769Crossref PubMed Scopus (48) Google Scholar), was also added using Modeler, and Cys415 was palmitoylated. Embedding the CB1 Receptor Model in a Lipid Bilayer—To build a receptor plus phospholipid bilayer system, we used a snapshot from a previous simulation of 1-palmitoyl-2-oleoylphosphatidylcholine (POPC). In these previous calculations, a patch of hydrated POPC was equilibrated for 3 ns at 310 K. The system was constructed using the VMD membrane and solvate plug-ins. The constant area/pressure NPAT ensemble was employed using a value of 68.0 Å2 as set via the VMD plug-in. This compares to earlier calculated values of 65.5 Å2 (30Shepherd C.M. Vogel H.J. Tieleman D.P. Biochem. 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The remaining system contained 240 POPC molecules, 19,737 water molecules, the CB1 receptor, and 11 chloride ions, the latter of which were added to achieve charge neutrality for this system, for a total of 96,074 atoms. Molecular Dynamics Simulations—The NAMD2 (35Kale L. Skeel R. Bhandaarkar M. Brunner R. Gursoy A. Krawetz N. Phillips J. Shinozaki A. Varadarajan K. Schulten K. J. Comp. Physics. 1999; 151: 283-312Crossref Scopus (2131) Google Scholar) molecular simulation package along with the CHARMM27 parameter set (36Feller S.E. MacKerell Jr., A.D. J. Phys. Chem. 2000; 104: 7510-7515Crossref Scopus (688) Google Scholar, 37MacKerell Jr., A.D. Bashford D. Bellot M. Dunbrack Jr., R.L. Evanseck J. Field M.J. Fischer S. Gao J. Guo H. Ha S. Joseph D. Kuchnir L. Kuczera K. Lau F.T.K. Mattos C. Michnick S. Ngo T. Nguyen D.T. Prodhom B. Reiher I.W.E. Roux B. Schlenkrich M. Smith J. Stote R. Straub J. Watanabe M. Wiorkiewicz-Kuczera J. Yin D. Karplus M. J. Phys. 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