Cloning and expression of the thyrotropin-releasing hormone receptor from GH3 rat anterior pituitary cells.
Functional thyrotropin-releasing hormone (TRH) receptors have been expressed in Xenopus laevis oocytes following the microinjection of total and poly(A)+ RNA from GH3 rat anterior pituitary tumour cells. Under voltage-clamp conditions, application of the peptide induced a biphasic Ca(2+)-dependent chloride current. The amplitude of the initial, fast, component of the response was dependent on the concentration of the hormone and on the amount of mRNA injected. Size fractionation of poly(A)+ RNA on a continuous sucrose gradient and Northern blot analysis indicated that the receptor was encoded by an mRNA of approx. 3.5 kb. A 3.28 kbp cDNA encoding the TRH receptor has been cloned and sequenced. Full functionality of the predicted 412-amino-acid receptor protein was demonstrated by functional expression of cell surface receptors in Xenopus oocytes after both cytoplasmic injection of sense RNA transcribed in vitro from this cDNA and nuclear injection of the cDNA under the control of the Herpes simplex virus thymidine kinase promoter. The predicted protein contains seven putative membrane-spanning domains and shows significant sequence identify with some G-protein-coupled receptors. RNA blot analysis indicates that the mRNA for the TRH receptor is exclusively expressed in the pituitary gland. Expression studies performed with clones in which the 3' region of the mRNA has been successively shortened indicate that the 3' terminal region is not an important determinant for efficient functional expression in oocytes.
de la Peña
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P
Departamento de Biología Funcional, Facultad de Medicina, Universidad de Oviedo, Spain.
Delgado
L M
LM
del Camino
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D
Barros
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F
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D002460:Cell Line; D003001:Cloning, Molecular / Q000379:methods; D004273:DNA, Neoplasm / Q000235:genetics; D015723:Gene Library; D051379:Mice; D008969:Molecular Sequence Data; D009865:Oocytes / Q000187:drug effects / Q000502:physiology*; D010903:Pituitary Gland, Anterior; D010911:Pituitary Neoplasms; D011061:Poly A / Q000235:genetics / Q000302:isolation & purification; D012313:RNA / Q000235:genetics / Q000302:isolation & purification; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012334:RNA, Neoplasm / Q000235:genetics / Q000302:isolation & purification; D051381:Rats; D017981:Receptors, Neurotransmitter / Q000187:drug effects / Q000235:genetics* / Q000502:physiology; D011985:Receptors, Serotonin / Q000187:drug effects / Q000235:genetics / Q000502:physiology; D018025:Receptors, Thyrotropin-Releasing Hormone; D011994:Recombinant Proteins / Q000187:drug effects / Q000378:metabolism; D012689:Sequence Homology, Nucleic Acid; D012701:Serotonin / Q000494:pharmacology; D013973:Thyrotropin-Releasing Hormone / Q000378:metabolism / Q000494:pharmacology*; D014158:Transcription, Genetic; D014982:Xenopus laevis
D004273:DNA, Neoplasm; D012333:RNA, Messenger; D012334:RNA, Neoplasm; D017981:Receptors, Neurotransmitter; D011985:Receptors, Serotonin; D018025:Receptors, Thyrotropin-Releasing Hormone; D011994:Recombinant Proteins; D011061:Poly A; D012701:Serotonin; D013973:Thyrotropin-Releasing Hormone; D012313:RNA