An efficient screening morphological test for the identification and characterization of cyclic AMP-coupled hormone receptors.
We describe here a new method for the identification of adenylate cyclase-regulating receptors using the morphological response of Y1 cells to cAMP. The efficiency of this method was demonstrated with a cloned beta 2-adrenergic receptor: the coding region of a genomic beta 2-adrenergic receptor clone was amplified using the polymerase chain reaction, so that in vitro RNA transcripts of this DNA could be obtained. Y1 cells were microinjected with this RNA and assayed for sensitivity to isoproterenol. In the presence of this agonist, the microinjected cells rapidly showed morphological changes identical to those observed in the presence of ACTH, forskolin, or cholera toxin, agents that enhance cAMP accumulation in the control uninjected cells. This suggests the integration, within the injected cell membrane, of a functional beta 2-adrenergic receptor, whose activation by isoproterenol resulted in the intracellular formation of cAMP. This new qualitative screening method for the identification of adenylate cyclase-regulating receptors can be extended to any unknown receptor coupled to adenylate cyclase and absent in these cells. It has been applied to the identification of the dog thyrotropin receptor.
Maenhaut
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C
Institute of Interdisciplinary Research, School of Medicine, Free University of Brussels, Belgium.
Libert
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F
D000310:Adrenal Gland Neoplasms; D000324:Adrenocorticotropic Hormone / Q000494:pharmacology; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D000242:Cyclic AMP / Q000502:physiology*; D006728:Hormones / Q000494:pharmacology; D008845:Microinjections; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000235:genetics; D011943:Receptors, Adrenergic, beta / Q000235:genetics; D011956:Receptors, Cell Surface / Q000187:drug effects / Q000235:genetics*; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured / Q000166:cytology / Q000187:drug effects / Q000378:metabolism
D006728:Hormones; D012333:RNA, Messenger; D011943:Receptors, Adrenergic, beta; D011956:Receptors, Cell Surface; D000324:Adrenocorticotropic Hormone; D000242:Cyclic AMP