CYTOPLASMIC SEQUENCES OF THE GROWTH-HORMONE RECEPTOR NECESSARY FOR SIGNAL-TRANSDUCTION

被引:92
作者
GOUJON, L
ALLEVATO, G
SIMONIN, G
PAQUEREAU, L
LECAM, A
CLARK, J
NIELSEN, JH
DJIANE, J
POSTELVINAY, MC
EDERY, M
KELLY, PA
机构
[1] HAGEDORN RES INST,DK-2820 GENTOFTE,DENMARK
[2] CTR PHARMACOL ENDOCRINOL,F-34094 MONTPELLIER,FRANCE
[3] INRA,F-78350 JOUY EN JOSAS,FRANCE
[4] INRA,UNITE ENDOCRINOL MOLEC,F-78350 JOUY EN JOSES,FRANCE
[5] AFRC,INST ANIM PHYSIOL & GENET RES,ROSLIN EH25 9PS,SCOTLAND
关键词
GROWTH HORMONE RECEPTOR MUTANTS; TRANSIENT TRANSFECTION; SERINE PROTEASE INHIBITOR; OVINE BETA-LACTOGLOBULIN; PROMOTER TESTS;
D O I
10.1073/pnas.91.3.957
中图分类号
O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];
学科分类号
07 ; 0710 ; 09 ;
摘要
To study structure-function relationships of the growth hormone (GH) receptor (GHR), two functional systems have been developed. CHO cells were transiently cotransfected with the cDNA encoding the full-length rat GHR and with a construct consisting of the 5' flanking region of one of two GH-dependent genes encoding ovine beta-lactoglobulin or serine protease inhibitor 2.1 (Spi 2.1, formerly Spi.1; the corresponding rat gene has recently been redesignated Spin2a) coupled to the bacterial reporter gene encoding chloramphenicol acetyltransferase (CAT). Transfected cells were grown in the absence and presence of human GH and dexamethasone for the Spi 2.1 gene construct. GH was able to activate each promoter (with almost-equal-to 4-fold induction of CAT activity) in a dose-dependent manner. For both tests, the maximal effect was observed at 20 nM human GH. These tests have been used to identify functional domains of the GHR. Two truncated (T) GHRs, lacking most or part of the cytoplasmic domain [called T276 (ending at residue 276) and T436 (ending at residue 436)], were unable to stimulate CAT activity. The GHR contains a proline-rich region, called ''Box I,'' conserved in the cytokine/GH/prolactin receptor family. Alanine substitutions for the four prolines of GHR Box I were introduced. Single proline-to-alanine mutations did not affect the functional activity of the GHR. However, modification of the four prolines together or deletion of the Box I (15 amino acids between positions 279 and 293) resulted in the complete absence of GH stimulation. Thus, the proline-rich region, shown to be important for other members of this receptor superfamily, is also critical for GH signal transduction.
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页码:957 / 961
页数:5
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