{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,12,11]],"date-time":"2025-12-11T20:07:30Z","timestamp":1765483650267,"version":"3.32.0"},"reference-count":55,"publisher":"Wiley","issue":"3","license":[{"start":{"date-parts":[[2005,7,25]],"date-time":"2005-07-25T00:00:00Z","timestamp":1122249600000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/onlinelibrary.wiley.com\/termsAndConditions#vor"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["The Journal of Peptide Research"],"published-print":{"date-parts":[[2005,9]]},"abstract":"<jats:p><jats:bold>Abstract:\u2002<\/jats:bold>The cannabinoid receptor 1 (CB1) cannabinoid receptor is an essential component of the cannabinergic system. It has been recognized as a therapeutic target for treating numerous diseases and is currently receiving considerable attention by the pharmaceutical community. Target\u2010based drug design, utilizing three\u2010dimensional information of receptor structure and ligand\u2010binding motifs, requires significant amounts of purified protein. To facilitate the purification of CB1, we have expressed the receptor fused to various epitope tags using the baculovirus expression system. In addition, expression levels and ligand\u2010binding profiles corresponding to the expressed fusion proteins have been compared. C\u2010terminal histidine (His)\u2010tagged CB1 gave a<jats:italic>B<\/jats:italic><jats:sub>max<\/jats:sub>higher than most other systems previously reported in the literature, and was selected for subsequent metal affinity chromatography purification and mass spectroscopic (MS) analysis. Moreover, cells expressing C\u2010terminal His\u2010tagged CB1 were shown to inhibit forskolin\u2010stimulated cyclic adenosine 3\u2032,5\u2032\u2010monophosphate (cAMP) production in a concentration\u2010dependent manner in the presence of CP\u201055,940, confirming the expressed receptor's functional characteristics. A Western blot analysis of the purified receptor showed several forms of CB1, the most abundant being a 57\u2003kDa monomeric protein. The purified CB1 preparations were subjected to protein digestion followed by MS. Fragments corresponding to &gt;70% of the receptor were identified by this method, confirming the identity and purity of the expressed protein. The work presented here demonstrates that epitope\u2010tagged CB1 can be expressed in sufficient amounts and purified to homogeneity for MS analysis. Moreover, these results will serve as a basis for future experiments aimed at characterizing the ligand\u2010binding domains using covalently reacting receptor probes.<\/jats:p>","DOI":"10.1111\/j.1399-3011.2005.00283.x","type":"journal-article","created":{"date-parts":[[2005,7,29]],"date-time":"2005-07-29T16:39:38Z","timestamp":1122655178000},"page":"138-150","source":"Crossref","is-referenced-by-count":26,"title":["Purification and mass spectroscopic analysis of human CB1 cannabinoid receptor functionally expressed using the baculovirus system"],"prefix":"10.1111","volume":"66","author":[{"given":"W.","family":"Xu","sequence":"first","affiliation":[]},{"given":"S.A.","family":"Filppula","sequence":"additional","affiliation":[]},{"given":"R.","family":"Mercier","sequence":"additional","affiliation":[]},{"given":"S.","family":"Yaddanapudi","sequence":"additional","affiliation":[]},{"given":"S.","family":"Pavlopoulos","sequence":"additional","affiliation":[]},{"given":"J.","family":"Cai","sequence":"additional","affiliation":[]},{"given":"W.M.","family":"Pierce","sequence":"additional","affiliation":[]},{"given":"A.","family":"Makriyannis","sequence":"additional","affiliation":[]}],"member":"311","published-online":{"date-parts":[[2008,12,5]]},"reference":[{"key":"e_1_2_7_2_2","doi-asserted-by":"publisher","DOI":"10.1038\/346561a0"},{"key":"e_1_2_7_3_2","doi-asserted-by":"publisher","DOI":"10.1038\/365061a0"},{"key":"e_1_2_7_4_2","doi-asserted-by":"publisher","DOI":"10.1038\/sj.bjp.0704327"},{"key":"e_1_2_7_5_2","doi-asserted-by":"publisher","DOI":"10.1016\/S0163-7258(02)00250-4"},{"key":"e_1_2_7_6_2","doi-asserted-by":"publisher","DOI":"10.2174\/1381612003399419"},{"key":"e_1_2_7_7_2","doi-asserted-by":"publisher","DOI":"10.1016\/S0163-7258(01)00130-9"},{"key":"e_1_2_7_8_2","doi-asserted-by":"publisher","DOI":"10.1016\/S1471-4892(01)00120-5"},{"key":"e_1_2_7_9_2","doi-asserted-by":"publisher","DOI":"10.1007\/BF03348205"},{"key":"e_1_2_7_10_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.pbb.2003.10.015"},{"key":"e_1_2_7_11_2","doi-asserted-by":"publisher","DOI":"10.1007\/s00520-003-0530-0"},{"key":"e_1_2_7_12_2","doi-asserted-by":"publisher","DOI":"10.1038\/nm0602-547"},{"key":"e_1_2_7_13_2","doi-asserted-by":"publisher","DOI":"10.1523\/JNEUROSCI.23-06-02453.2003"},{"key":"e_1_2_7_14_2","doi-asserted-by":"publisher","DOI":"10.1126\/science.289.5480.739"},{"key":"e_1_2_7_15_2","doi-asserted-by":"publisher","DOI":"10.1146\/annurev.pa.35.040195.003135"},{"key":"e_1_2_7_16_2","doi-asserted-by":"crossref","first-page":"834","DOI":"10.1016\/S0026-895X(24)12547-3","article-title":"The difference between the CB1 and CB2 cannabinoid receptors at position 5.46 is crucial for the selectivity of WIN55212\u20102 for CB2","volume":"56","author":"Song Z.H.","year":"1999","journal-title":"Mol. Pharmacol."},{"key":"e_1_2_7_17_2","doi-asserted-by":"publisher","DOI":"10.1016\/S0006-2952(98)00081-1"},{"key":"e_1_2_7_18_2","doi-asserted-by":"publisher","DOI":"10.1023\/A:1018383808401"},{"key":"e_1_2_7_19_2","doi-asserted-by":"crossref","first-page":"605","DOI":"10.1016\/S0026-895X(24)12187-6","article-title":"Role of a conserved lysine residue in the peripheral cannabinoid receptor (CB2): evidence for subtype specificity","volume":"55","author":"Tao Q.","year":"1999","journal-title":"Mol. Pharmacol."},{"key":"e_1_2_7_20_2","doi-asserted-by":"publisher","DOI":"10.1111\/j.1399-3011.2004.00188.x"},{"key":"e_1_2_7_21_2","doi-asserted-by":"publisher","DOI":"10.1124\/mol.64.3.570"},{"key":"e_1_2_7_22_2","doi-asserted-by":"publisher","DOI":"10.1016\/S0014-5793(01)02711-9"},{"volume-title":"Current Protocols in Molecular Biology","year":"1987","author":"Ausubel F.M.","key":"e_1_2_7_23_2"},{"key":"e_1_2_7_24_2","first-page":"513","article-title":"Sample preparation methods for mass spectrometric peptide mapping directly from 2\u2010DE gels","volume":"112","author":"Jensen O.N.","year":"1999","journal-title":"Methods Mol. Biol."},{"key":"e_1_2_7_25_2","first-page":"651","article-title":"Mutation of a highly conserved aspartate residue in the second transmembrane domain of the cannabinoid receptors, CB1 and CB2, disrupts G\u2010protein coupling","volume":"285","author":"Tao Q.","year":"1998","journal-title":"J. Pharmacol. Exp. Ther."},{"key":"e_1_2_7_26_2","doi-asserted-by":"publisher","DOI":"10.1046\/j.1471-4159.1998.70010366.x"},{"key":"e_1_2_7_27_2","doi-asserted-by":"publisher","DOI":"10.1111\/j.1471-4159.1990.tb08815.x"},{"key":"e_1_2_7_28_2","doi-asserted-by":"publisher","DOI":"10.1111\/j.1471-4159.1991.tb06379.x"},{"key":"e_1_2_7_29_2","doi-asserted-by":"publisher","DOI":"10.1016\/0006-2952(94)90134-1"},{"key":"e_1_2_7_30_2","doi-asserted-by":"publisher","DOI":"10.1111\/j.1471-4159.1993.tb03576.x"},{"key":"e_1_2_7_31_2","doi-asserted-by":"publisher","DOI":"10.1042\/bj3120637"},{"key":"e_1_2_7_32_2","doi-asserted-by":"publisher","DOI":"10.1042\/bj2790129"},{"key":"e_1_2_7_33_2","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.90.16.7656"},{"key":"e_1_2_7_34_2","first-page":"891","article-title":"A lysine residue of the cannabinoid receptor is critical for receptor recognition by several agonists but not WIN55212\u20102","volume":"49","author":"Song Z.H.","year":"1996","journal-title":"Mol. Pharmacol."},{"key":"e_1_2_7_35_2","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.271.12.6941"},{"volume-title":"Pharmacologic Analysis of Drug\u2010receptor Interaction","year":"1993","author":"Kenakin T.P.","key":"e_1_2_7_36_2"},{"key":"e_1_2_7_37_2","doi-asserted-by":"publisher","DOI":"10.1091\/mbc.9.8.2231"},{"key":"e_1_2_7_38_2","doi-asserted-by":"publisher","DOI":"10.1210\/jc.2003-031047"},{"key":"e_1_2_7_39_2","doi-asserted-by":"publisher","DOI":"10.1017\/S0033583500003504"},{"key":"e_1_2_7_40_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.lfs.2003.09.005"},{"key":"e_1_2_7_41_2","doi-asserted-by":"publisher","DOI":"10.1016\/S0165-6147(00)01799-5"},{"key":"e_1_2_7_42_2","doi-asserted-by":"publisher","DOI":"10.1038\/35067575"},{"key":"e_1_2_7_43_2","doi-asserted-by":"publisher","DOI":"10.1016\/S0009-3084(02)00151-2"},{"key":"e_1_2_7_44_2","doi-asserted-by":"publisher","DOI":"10.1016\/S0006-2952(02)01031-6"},{"key":"e_1_2_7_45_2","first-page":"837","article-title":"The third transmembrane helix of the cannabinoid receptor plays a role in the selectivity of aminoalkylindoles for CB2, peripheral cannabinoid receptor","volume":"291","author":"Chin C.N.","year":"1999","journal-title":"J. Pharmacol. Exp. Ther."},{"key":"e_1_2_7_46_2","doi-asserted-by":"publisher","DOI":"10.1046\/j.1471-4159.1999.0722032.x"},{"key":"e_1_2_7_47_2","doi-asserted-by":"publisher","DOI":"10.1016\/0376-8716(94)90006-X"},{"key":"e_1_2_7_48_2","first-page":"1172","article-title":"Evaluation of cannabinoid receptor binding and in vivo activities for anandamide analogs","volume":"273","author":"Adams I.B.","year":"1995","journal-title":"J. Pharmacol. Exp. Ther."},{"key":"e_1_2_7_49_2","first-page":"605","article-title":"Determination and characterization of a cannabinoid receptor in rat brain","volume":"34","author":"Devane W.A.","year":"1988","journal-title":"Mol. Pharmacol."},{"key":"e_1_2_7_50_2","first-page":"838","article-title":"Cannabinoid agonists stimulate both receptor\u2010 and non\u2010receptor\u2010mediated signal transduction pathways in cells transfected with and expressing cannabinoid receptor clones","volume":"42","author":"Felder C.C.","year":"1992","journal-title":"Mol. Pharmacol."},{"key":"e_1_2_7_51_2","doi-asserted-by":"publisher","DOI":"10.1523\/JNEUROSCI.11-02-00563.1991"},{"key":"e_1_2_7_52_2","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.87.5.1932"},{"key":"e_1_2_7_53_2","doi-asserted-by":"publisher","DOI":"10.1016\/0091-3057(93)90546-6"},{"key":"e_1_2_7_54_2","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.270.23.13973"},{"key":"e_1_2_7_55_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.neuropharm.2005.11.008"},{"key":"e_1_2_7_56_2","doi-asserted-by":"publisher","DOI":"10.1034\/j.1399-3011.2002.21066.x"}],"container-title":["The Journal of Peptide Research"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/api.wiley.com\/onlinelibrary\/tdm\/v1\/articles\/10.1111%2Fj.1399-3011.2005.00283.x","content-type":"unspecified","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/onlinelibrary.wiley.com\/doi\/pdf\/10.1111\/j.1399-3011.2005.00283.x","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,1,2]],"date-time":"2025-01-02T19:51:48Z","timestamp":1735847508000},"score":1,"resource":{"primary":{"URL":"https:\/\/onlinelibrary.wiley.com\/doi\/10.1111\/j.1399-3011.2005.00283.x"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2005,9]]},"references-count":55,"journal-issue":{"issue":"3","published-print":{"date-parts":[[2005,9]]}},"alternative-id":["10.1111\/j.1399-3011.2005.00283.x"],"URL":"https:\/\/doi.org\/10.1111\/j.1399-3011.2005.00283.x","archive":["Portico"],"relation":{},"ISSN":["1397-002X"],"issn-type":[{"type":"print","value":"1397-002X"}],"subject":[],"published":{"date-parts":[[2005,9]]}}}