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High level of expression of functional human platelet alpha 2-adrenergic receptors in a stable mouse C127 cell line.
Bresnahan, M R; Flordellis, C S; Vassilatis, D K; Makrides, S C; Zannis, V I; Gavras, H.
Afiliação
  • Bresnahan MR; Section of Hypertension and Atherosclerosis, Boston University School of Medicine, MA 02118.
Biochim Biophys Acta ; 1052(3): 439-45, 1990 May 22.
Article em En | MEDLINE | ID: mdl-1972337
ABSTRACT
We have generated, by transfection and proper selection, a stable mouse C127 cell line which expresses the human alpha 2-adrenergic receptor gene. The size of the mRNA produced by the cloned gene is 1.8 kb. Electrophoretic analysis and autoradiography of cell membrane proteins photoaffinity labeled with p-[3H]azidoclonidine gave a broad protein band of molecular mass of approx. 64 kDa. Saturation binding with [3H]rauwolscine as ligand gave an equilibrium dissociation constant of 1.29 +/- 0.46 nM (mean +/- S.D.) and binding capacity range of 18-35 pmol/mg membrane protein, with (3-6) x 10(6) receptors per cell. Antagonist competition experiments displayed the order of potency yohimbine greater than rauwolscine greater than phentolamine much greater than prazosin. Agonist competitions demonstrated the order of potency p-aminoclonidine greater than (-)epinephrine much greater than (+)epinephrine much greater than (-)isoproterenol. This pharmacological profile is characteristic of the human platelet alpha 2-adrenergic receptor. The expressed receptor is able to couple to the Gi protein. Thus, when epinephrine competition for specific binding of [3H]rauwolscine was performed in the presence of 1 mM MgCl2, 1 mM Gpp[NH]p increased the Ki for epinephrine from 164 to 315 nM. Following preincubation of cultures with 1 mM isobutylmethylxanthine, 1 microM epinephrine decreased forskolin-stimulated cellular cyclic AMP accumulation by 72%. The response was biphasic, and the attenuation effect disappeared at 100 microM epinephrine. A transfected clone which did not demonstrate detectable alpha 2-adrenergic receptor mRNA displayed low levels of alpha 2-adrenergic receptor, (less than 50 fmol/mg membrane protein), similar to those found in the parent C127 cell line. In this clone, epinephrine did not attenuate but, rather, enhanced forskolin-stimulated cyclic AMP accumulation. This new C127 cell line expressing high levels of alpha 2-adrenergic receptor provides an abundant source of a single human adrenergic receptor subtype in membrane-bound conformation which is able to couple to the Gi protein and inhibit forskolin-stimulated adenylate cyclase activity. This cell line will facilitate studies of the structure function relationship of the alpha 2-adrenergic receptor and should aid in separating the components of various signal transduction mechanisms putatively attributed to this receptor.
Assuntos
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Coleções: 01-internacional Base de dados: MEDLINE Assunto principal: Plaquetas / Expressão Gênica / Receptores Adrenérgicos alfa Limite: Animals / Humans Idioma: En Ano de publicação: 1990 Tipo de documento: Article
Buscar no Google
Coleções: 01-internacional Base de dados: MEDLINE Assunto principal: Plaquetas / Expressão Gênica / Receptores Adrenérgicos alfa Limite: Animals / Humans Idioma: En Ano de publicação: 1990 Tipo de documento: Article