RESUMEN
A yeast two-hybrid screen, using the carboxyl tail of the human mu opioid receptor as bait and a human brain cDNA library as target, indicated that the carboxyl terminal portion of hlj1, a member of the human heat shock protein 40 family, interacts with the carboxyl tail of the human mu opioid receptor. To determine if direct in vitro binding occurs between these two proteins, we performed overlay experiments. Results from the overlay experiments showed that binding occurs between the His fusion protein of hlj1 and the GST fusion protein of the carboxyl tail of the human mu opioid receptor. In contrast, no binding with the His fusion protein of hlj1 occurred with GST alone or the GST fusion protein of the third cytoplasmic loop of the human mu opioid receptor. Results from co-immunoprecipitation studies, carried out in whole HEK cell lysates, confirmed in vivo binding between these two proteins. Immunofluorescent studies, using laser scanning confocal microscopy, showed significant co-localization between hlj1 and the human mu opioid receptor in the cell membrane. The function of this protein-protein interaction and its physiological relevance in animal and human brain is yet to be determined.
Asunto(s)
Proteínas del Choque Térmico HSP40/metabolismo , Receptores Opioides mu/metabolismo , Sitios de Unión/fisiología , Western Blotting/métodos , Línea Celular , Humanos , Inmunohistoquímica/métodos , Inmunoprecipitación/métodos , Microscopía Confocal/métodos , Mutagénesis/fisiología , Unión Proteica , Mapeo de Interacción de Proteínas/métodos , Receptores Opioides mu/química , Transfección/métodos , Técnicas del Sistema de Dos HíbridosRESUMEN
The cDNA coding for the bovine mu-opioid receptor has been cloned and sequenced. Conserved sequences from murine delta-receptor cDNA were used as primers in polymerase chain reaction (PCR) to amplify cDNA, prepared by reverse transcription of bovine brain mRNA. This cDNA was used to probe a bovine brain library. The partial sequence obtained was extended to provide the full length clone by PCR. The cDNA has an open reading frame of 1203 base pairs (bp) with a 3'-untranslated region of 1900 bp and a 5'-untranslated region of 265 bp. The protein contains 401 amino acids and has 94% amino acid identity with the human and 91% with the rat mu-opioid receptor. It has the putative seven transmembrane domains, characteristic of G protein-coupled receptors and contains 5 potential N-linked glycosylation sites near the N-terminus. Several potential phosphorylation sites and a putative palmitoylation site are also present. The receptor was stably expressed in HEK293 cells. The binding profile was found to be that of a typical mu receptor, i. e., mu agonists and antagonists, but not delta and kappa ligands, bound with high affinity. Functional assays, namely, opioid stimulation of [35S]GTPgammaS binding and inhibition of forskolin-activated adenylyl cyclase, were also found to be highly specific for mu-opioid agonists. The receptor was downregulated by chronic exposure to mu agonists but not delta or kappa agonists. Evidence is presented indicating that the cloned receptor is the same as the bovine mu receptor previously purified to homogeneity in our laboratory. No evidence was found for genes for multiple mu-type opioid receptors.
Asunto(s)
Bencenoacetamidas , Receptores Opioides mu/genética , Adenilil Ciclasas/efectos de los fármacos , Adenilil Ciclasas/metabolismo , Secuencia de Aminoácidos , Analgésicos Opioides/metabolismo , Analgésicos Opioides/farmacología , Animales , Secuencia de Bases , Unión Competitiva/efectos de los fármacos , Bovinos , Línea Celular , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Clonación Molecular , Colforsina/farmacología , Cuerpo Estriado/química , ADN Complementario/química , ADN Complementario/genética , Diprenorfina/metabolismo , Diprenorfina/farmacología , Relación Dosis-Respuesta a Droga , Encefalina Ala(2)-MeFe(4)-Gli(5)/metabolismo , Encefalina Ala(2)-MeFe(4)-Gli(5)/farmacología , Encefalina D-Penicilamina (2,5)/metabolismo , Encefalina D-Penicilamina (2,5)/farmacología , Expresión Génica , Regulación de la Expresión Génica/efectos de los fármacos , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Humanos , Datos de Secuencia Molecular , Pirrolidinas/metabolismo , Pirrolidinas/farmacología , Ensayo de Unión Radioligante , Receptores Opioides mu/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Alineación de Secuencia , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Radioisótopos de Azufre , TritioRESUMEN
We have investigated the role of cysteine residues in a highly purified mu opioid receptor protein (muORP) by examining the effect of -SH reagents on the binding of opioid ligands. Treatment of muORP, which is devoid of additional proteins, eliminates complications that arise from reaction of -SH reagents with other components, such as G proteins. Reagents tested include N-ethylmaleimide, 5,5'-dithiobis(2-nitrobenzoic) acid, and two derivatives of methanethiosulfonate. Specific opioid binding was inactivated by micromolar concentrations of all -SH reagents tested. Agonist binding ([3H]DAMGO) was much more sensitive to inactivation than antagonist binding ([3H]bremazocine). Prebinding muORP with 100 nM naloxone protected antagonist and agonist binding from inactivation by -SH reagents. The results of these experiments strongly suggest that at least one, and possibly more, reactive cysteine residue(s) is present on the mu opioid receptor protein molecule, positioned near the ligand binding site and accessible to -SH reagents.