Cytoplasmic sequences of the growth hormone receptor necessary for signal transduction

TY - JOUR

T1 - Cytoplasmic sequences of the growth hormone receptor necessary for signal transduction

AU - Goujon, L

AU - Allevato, G

AU - Simonin, G

AU - Paquereau, L

AU - Le Cam, A

AU - Clark, J

AU - Djiane, J

AU - Postel-Vinay, M C

AU - Nielsen, Jens Høiriis

AU - Edery, M

PY - 1994/2/1

Y1 - 1994/2/1

N2 - 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 approximately 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.

AB - 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 approximately 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.

KW - Amino Acid Sequence

KW - Animals

KW - Base Sequence

KW - CHO Cells

KW - Chloramphenicol O-Acetyltransferase

KW - Cricetinae

KW - Cytoplasm

KW - DNA, Complementary

KW - Genes, Reporter

KW - Growth Hormone

KW - Humans

KW - Lactoglobulins

KW - Molecular Sequence Data

KW - Promoter Regions, Genetic

KW - Rats

KW - Receptors, Somatotropin

KW - Sequence Deletion

KW - Sheep

KW - Signal Transduction

KW - Transcription, Genetic

KW - Transfection

M3 - Journal article

C2 - 8302873

SN - 0027-8424

VL - 91

SP - 957

EP - 961

JO - Proceedings of the National Academy of Sciences of the United States of America

JF - Proceedings of the National Academy of Sciences of the United States of America

IS - 3

ER -