Cloning of a cDNA encoding an ectoenzyme that degrades thyrotropin-releasing hormone.
Thyrotropin-releasing hormone (TRH) is an important extracellular signal substance that acts as a hypothalamic-releasing factor, which stimulates the release of adenohypophyseal hormones and functions as a neurotransmitter/neuromodulator in the central and peripheral nervous system. The inactivation of TRH after its release is catalyzed by an ectoenzyme localized preferentially on neuronal cells in the brain and on lactotrophic pituitary cells. This enzyme exhibits a very high degree of substrate specificity as well as other unusual properties. The activity of the adenohypophyseal enzyme is stringently controlled by estradiol and thyroid hormones, indicating that this enzyme itself may serve regulatory functions. Fragments of the enzyme isolated from rat or pig brain were generated by enzymatic digestion or cyanogen bromide cleavage, purified by reverse-phase HPLC, and sequenced. PCR amplification and screening of cDNA libraries from rat brain and pituitary led to the identification and isolation of a cDNA that encodes a protein of 1025 amino acids. The analysis of the deduced amino acid sequence was consistent with the identification of the enzyme as a glycosylated, membrane-anchored Zn metallopeptidase. Furthermore, Northern blot analysis demonstrated that the mRNA levels paralleled the tissue distribution of the enzyme and that in pituitary tissue the transcript levels rapidly increased when the animals were treated with triiodothyronine. Finally, transient transfection of COS-7 cells with this cDNA led to the expression of an active ectopeptidase that displayed the characteristics of the TRH-degrading ectoenzyme.
Schauder
B
B
Max-Planck-Institut für Experimentelle Endokrinologie, Hannover, Federal Republic of Germany.
Schomburg
L
L
Köhrle
J
J
Bauer
K
K
D000595:Amino Acid Sequence; D000626:Aminopeptidases / Q000096:biosynthesis* / Q000737:chemistry / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D001921:Brain / Q000201:enzymology*; D002460:Cell Line; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000378:metabolism; D007668:Kidney / Q000201:enzymology; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D008168:Lung / Q000201:enzymology; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D016133:Polymerase Chain Reaction; D011487:Protein Conformation; D011761:Pyrrolidonecarboxylic Acid / Q000031:analogs & derivatives; D051381:Rats; D017207:Rats, Sprague-Dawley; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013552:Swine; D013973:Thyrotropin-Releasing Hormone / Q000378:metabolism; D014162:Transfection
D018076:DNA, Complementary; D010446:Peptide Fragments; D011994:Recombinant Proteins; D013973:Thyrotropin-Releasing Hormone; D000626:Aminopeptidases; C023166:pyroglutamyl-peptidase II; D011761:Pyrrolidonecarboxylic Acid