{"corpus_id":17639003,"paper_sha":"134efc44c16af671a8557e657e7012c6351a2881","doi":"10.1016/s0026-895x(24)26450-6","arxiv_id":null,"pmid":10617687,"pmcid":null,"mag_id":1893098545,"dblp_id":null,"acl_id":null,"title":"Use of constitutive G protein-coupled receptor activity for drug discovery.","year":2000,"publication_date":"2000-01-01","venue":"Molecular Pharmacology","journal":{"name":"Molecular pharmacology","pages":"\n          125-34\n        ","volume":"57 1"},"journal_issn":null,"journal_title":null,"publication_types":["JournalArticle"],"pubmed_pub_types":["Journal Article"],"s2_fields_of_study":["Biology","Medicine","Chemistry"],"reference_count":29,"citation_count":103,"influential_citation_count":4,"is_open_access":false,"arxiv_categories":null,"arxiv_license":null,"arxiv_journal_ref":null,"mesh_headings":[{"d":"Amino Acid Sequence","mj":false,"ui":"D000595"},{"d":"Animals","mj":false,"ui":"D000818"},{"d":"Calcitonin","mj":false,"qs":[{"q":"analogs & derivatives","mj":false,"ui":"Q000031"},{"q":"pharmacology","mj":false,"ui":"Q000494"}],"ui":"D002116"},{"d":"Drug Evaluation, Preclinical","mj":true,"ui":"D004353"},{"d":"GTP-Binding Protein alpha Subunits, Gi-Go","mj":false,"qs":[{"q":"metabolism","mj":false,"ui":"Q000378"}],"ui":"D019206"},{"d":"GTP-Binding Protein alpha Subunits, Gs","mj":false,"qs":[{"q":"metabolism","mj":false,"ui":"Q000378"}],"ui":"D019205"},{"d":"GTP-Binding Proteins","mj":false,"qs":[{"q":"metabolism","mj":true,"ui":"Q000378"}],"ui":"D019204"},{"d":"Humans","mj":false,"ui":"D006801"},{"d":"Ligands","mj":false,"ui":"D008024"},{"d":"Melanophores","mj":false,"qs":[{"q":"drug effects","mj":false,"ui":"Q000187"},{"q":"metabolism","mj":false,"ui":"Q000378"}],"ui":"D008547"},{"d":"Melanosomes","mj":false,"qs":[{"q":"drug effects","mj":false,"ui":"Q000187"},{"q":"metabolism","mj":false,"ui":"Q000378"}],"ui":"D020460"},{"d":"Models, Chemical","mj":false,"ui":"D008956"},{"d":"Molecular Sequence Data","mj":false,"ui":"D008969"},{"d":"Protein Conformation","mj":false,"ui":"D011487"},{"d":"Receptors, Calcitonin","mj":false,"qs":[{"q":"agonists","mj":false,"ui":"Q000819"},{"q":"chemistry","mj":false,"ui":"Q000737"},{"q":"genetics","mj":false,"ui":"Q000235"},{"q":"metabolism","mj":true,"ui":"Q000378"}],"ui":"D018003"},{"d":"Receptors, Cell Surface","mj":false,"qs":[{"q":"agonists","mj":false,"ui":"Q000819"},{"q":"chemistry","mj":false,"ui":"Q000737"},{"q":"genetics","mj":false,"ui":"Q000235"},{"q":"metabolism","mj":false,"ui":"Q000378"}],"ui":"D011956"},{"d":"Sequence Homology, Amino Acid","mj":false,"ui":"D017386"},{"d":"Transfection","mj":false,"ui":"D014162"},{"d":"Xenopus laevis","mj":false,"ui":"D014982"}],"chemicals":[{"n":"Ligands","ui":"D008024","reg":"0"},{"n":"Receptors, Calcitonin","ui":"D018003","reg":"0"},{"n":"Receptors, Cell Surface","ui":"D011956","reg":"0"},{"n":"Calcitonin","ui":"D002116","reg":"9007-12-9"},{"n":"GTP-Binding Proteins","ui":"D019204","reg":"EC 3.6.1.-"},{"n":"GTP-Binding Protein alpha Subunits, Gi-Go","ui":"D019206","reg":"EC 3.6.5.1"},{"n":"GTP-Binding Protein alpha Subunits, Gs","ui":"D019205","reg":"EC 3.6.5.1"}],"comments_corrections":null,"source_flags":5,"s2_open_access_pdf_url":null,"s2_open_access_landing_url":null,"s2_open_access_license":null,"s2_open_access_status":null,"pmc_open_access_pdf_url":null,"pmc_open_access_landing_url":null,"pmc_open_access_license":null,"pmc_open_access_status":null,"unpaywall_open_access_pdf_url":null,"unpaywall_open_access_landing_url":null,"unpaywall_open_access_license":null,"unpaywall_open_access_status":null,"abstract":"This article describes the behavior of transiently transfected human receptors into melanophores and the potential use of constitutive receptor activity to screen for new drug entities. Specifically, transient transfection of melanophores with different concentrations of receptor cDNA presumably leads to increased levels of receptor expression. This leads to an increased response to agonists (both maxima and potency) and, in some cases, an agonist-independent constitutive receptor activity. Transfections with increasing concentrations of the G(s) protein-coupled human calcitonin receptor type 2 (hCTR2) cDNA produced sufficient levels of constitutively activated receptor to cause elevated basal cellular responses. This was observed as a decrease in the transmittance of light through melanophores (consistent with G(s) protein activation) and increased response to human calcitonin. The receptor-mediated nature of this response was confirmed by its reversal with the hCTR2 peptide inverse agonist AC512. A collection of ligands for hCTR2 either increased or decreased constitutive hCTR2 activity, suggesting that the constitutive system was a sensitive discriminator of positive and negative ligand efficacy. Similar results were obtained with G(i)-protein-coupled receptors. Transient transfection of NPY1, NPY2, NPY4, CXCR4, and CCR5 cDNA produced increased light transmittance through melanophores (consistent with G(i)-protein activation). NPY1 cDNA produced little constitutive response on transfection, whereas maximal levels of constitutive activity ranging from 30 to 45% were observed for the other G(i)-protein-coupled receptors. Responses to agonists for these receptors increased (both maxima and potency) with increasing cDNA transfection. The receptor/G(i)-protein nature of both the constitutive and agonist-mediated responses was confirmed by elimination with pertussis toxin pretreatment. These data are discussed in terms of the theoretical aspects of constitutive receptor activity and the applicability of this approach for the general screening of G protein-coupled orphan receptors.","claims":[{"public_id":"cl_c0f45d6ae806a3e49419b487c0b80a23","status":"active","text":"Constitutive receptor activity in the melanophore assay is applicable as a general screening approach for G protein-coupled orphan receptors.","confidence":0.9,"contributors":[{"id":1,"public_id":"12632b8b5f","public_label":"Anonymous (12632b8b5f)","roles":["extraction"],"url":"https://sah.borca.ai/u/12632b8b5f"}],"url":"https://sah.borca.ai/claims/cl_c0f45d6ae806a3e49419b487c0b80a23"},{"public_id":"cl_684ef589c9b84c41fff008af49918aab","status":"active","text":"For several G(i)-coupled receptors, transfection produced constitutive activity of about 30% to 45% for NPY2, NPY4, CXCR4, and CCR5, while NPY1 showed little constitutive response; agonist maxima and potency increased with cDNA transfection, and pertussis toxin eliminated both constitutive and agonist-mediated responses.","confidence":0.96,"contributors":[{"id":1,"public_id":"12632b8b5f","public_label":"Anonymous (12632b8b5f)","roles":["extraction"],"url":"https://sah.borca.ai/u/12632b8b5f"}],"url":"https://sah.borca.ai/claims/cl_684ef589c9b84c41fff008af49918aab"},{"public_id":"cl_22bf856741460bdbcd60a587c8ec35aa","status":"active","text":"Increasing hCTR2 cDNA expression produced constitutively activated receptor levels sufficient to lower light transmittance and enhance the response to human calcitonin, and the effect was reversed by the hCTR2 inverse agonist AC512.","confidence":0.99,"contributors":[{"id":1,"public_id":"12632b8b5f","public_label":"Anonymous (12632b8b5f)","roles":["extraction"],"url":"https://sah.borca.ai/u/12632b8b5f"}],"url":"https://sah.borca.ai/claims/cl_22bf856741460bdbcd60a587c8ec35aa"},{"public_id":"cl_d457ee8a692f657ab32a4a46a740519d","status":"active","text":"The constitutive hCTR2 system discriminated positive and negative ligand efficacy, because different hCTR2 ligands either increased or decreased basal activity.","confidence":0.95,"contributors":[{"id":1,"public_id":"12632b8b5f","public_label":"Anonymous (12632b8b5f)","roles":["extraction"],"url":"https://sah.borca.ai/u/12632b8b5f"}],"url":"https://sah.borca.ai/claims/cl_d457ee8a692f657ab32a4a46a740519d"},{"public_id":"cl_c9e6ffa004b13b9fbeed73b50a8ef9ab","status":"active","text":"Transient transfection of melanophores with increasing receptor cDNA can generate elevated basal signaling from constitutively active receptors and stronger agonist responses.","confidence":0.97,"contributors":[{"id":1,"public_id":"12632b8b5f","public_label":"Anonymous (12632b8b5f)","roles":["extraction"],"url":"https://sah.borca.ai/u/12632b8b5f"}],"url":"https://sah.borca.ai/claims/cl_c9e6ffa004b13b9fbeed73b50a8ef9ab"}],"concepts":[{"public_id":"co_0adfae5a33966e0483ebf1dc640a69d1","status":"active","name":"AC512","description":"A 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occurs in the absence of an added agonist.","types":["phenomenon"],"aliases":["constitutive activity"],"contributors":[{"id":1,"public_id":"12632b8b5f","public_label":"Anonymous (12632b8b5f)","roles":["extraction"],"url":"https://sah.borca.ai/u/12632b8b5f"}],"url":"https://sah.borca.ai/concepts/co_3ede6e0f647dcbed89bbf738eddee134"},{"public_id":"co_6fb8d94c2efc5dfa0e0f09d7efd0ddb0","status":"active","name":"G(i)-protein-coupled receptors","description":"Receptors that signal through inhibitory G proteins and were tested here for constitutive activity.","types":["receptor family"],"aliases":["Gi-coupled receptors"],"contributors":[{"id":1,"public_id":"12632b8b5f","public_label":"Anonymous (12632b8b5f)","roles":["extraction"],"url":"https://sah.borca.ai/u/12632b8b5f"}],"url":"https://sah.borca.ai/concepts/co_6fb8d94c2efc5dfa0e0f09d7efd0ddb0"},{"public_id":"co_94f674a969e47b77c8a12bbe8073952f","status":"active","name":"transient transfection","description":"Short-term introduction of cDNA into cells to express receptor proteins.","types":["method"],"aliases":[],"contributors":[{"id":1,"public_id":"12632b8b5f","public_label":"Anonymous (12632b8b5f)","roles":["extraction"],"url":"https://sah.borca.ai/u/12632b8b5f"}],"url":"https://sah.borca.ai/concepts/co_94f674a969e47b77c8a12bbe8073952f"},{"public_id":"co_968605e8bc1e6026ba9eac6afd6a23b1","status":"active","name":"melanophores","description":"Pigment cells used here as a cellular assay system for monitoring receptor-mediated light transmittance changes.","types":["cell type","assay system"],"aliases":[],"contributors":[{"id":1,"public_id":"12632b8b5f","public_label":"Anonymous (12632b8b5f)","roles":["extraction"],"url":"https://sah.borca.ai/u/12632b8b5f"}],"url":"https://sah.borca.ai/concepts/co_968605e8bc1e6026ba9eac6afd6a23b1"},{"public_id":"co_a2b0a1e9a1b594b699b3120a0c2de2d3","status":"active","name":"G protein-coupled orphan receptors","description":"G protein-coupled receptors with unknown endogenous ligands that 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