Molecular cloning of a complementary deoxyribonucleic acid encoding the thyrotropin-releasing hormone receptor and regulation of its messenger ribonucleic acid in rat GH cells.
Rat pituitary GH cells have been used extensively to study the biochemical actions of TRH on lactotropic cells. To investigate the structure and regulation of the rat TRH receptor (rTRHR), we have cloned its cDNA from GH4C1 cells. Using the polymerase chain reaction with degenerate primers and pools of cloned cDNAs from a GH4C1 cDNA library, a fragment sharing high similarity to the mouse thyrotrope TRHR (mTRHR) was identified. Conventional library screening with this fragment was used to isolate a single cDNA. mRNA synthesized in vitro from this cDNA was injected into Xenopus oocytes, and a characteristic conductance response to TRH was detected by voltage clamp recording. DNA sequence analysis revealed a molecule of 412 amino acid residues, with 96% similarity to the mTRHR. However, in contrast to the mTRHR, the rTRHR had an additional 19 amino acid residues at its carboxy-terminus. A mRNA of about 4 kilobases was identified in GH3 cells. Regulation of the rTRHR mRNA concentration was studied in GH3 cells. Steady state rTRHR mRNA levels were decreased to 30% of the control level by incubation with TRH for 48 h and increased 4-fold by incubation with dexamethasone for 12 h. Southern blot analysis of genomic DNA from GH3 cells gave a simple banding pattern consistent with a single copy gene. We conclude that the rTRHR shares high primary sequence similarity to the mTRHR, but the rTRHR has an extension of 19 amino acids at its carboxy-terminus, which is lacking in the mTRHR.
Zhao
D
D
Department of Molecular and Cellular Toxicology, Harvard School of Public Health, Boston, Massachusetts 02115.
Yang
J
J
Jones
K E
KE
Gerald
C
C
Suzuki
Y
Y
Hogan
P G
PG
Chin
W W
WW
Tashjian
A H
AH
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015139:Blotting, Southern; D002460:Cell Line; D003001:Cloning, Molecular; D004273:DNA, Neoplasm / Q000235:genetics / Q000302:isolation & purification; D015972:Gene Expression Regulation, Neoplastic; D015723:Gene Library; D051379:Mice; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D009865:Oocytes / Q000502:physiology; D010911:Pituitary Neoplasms; D016133:Polymerase Chain Reaction / Q000379:methods; D012333:RNA, Messenger / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D051381:Rats; D017981:Receptors, Neurotransmitter / Q000235:genetics* / Q000378:metabolism; D018025:Receptors, Thyrotropin-Releasing Hormone; D015183:Restriction Mapping; D012689:Sequence Homology, Nucleic Acid; D013973:Thyrotropin-Releasing Hormone / Q000378:metabolism; D014982:Xenopus laevis
D004273:DNA, Neoplasm; D009838:Oligodeoxyribonucleotides; D012333:RNA, Messenger; D017981:Receptors, Neurotransmitter; D018025:Receptors, Thyrotropin-Releasing Hormone; D013973:Thyrotropin-Releasing Hormone