Functional
Expression of Guinea Pig Growth Hormone Receptor
and Its Mutants in Mammalian Cells
LIAO Zhi-Yong, ZHANG Xin-Na,
JING Nai-He, ZHU Shang-Quan*
( Key Laboratory of Proteomics, Institute of Biochemistry
and Cell Biology, Shanghai Institutes for Biological Sciences, the Chinese
Academy of Sciences, Shanghai 200031, China )
Abstract The cDNA of guinea pig (Cavia
porcellus) growth hormone receptor (gpGHR) was cloned using RT-PCR in our
laboratory. By sequence alignment, substitutions of amino acids conserved in
other mammalian GHRs were found. For example, histidine-168 and tyrosine-332
equivalent to positions 170 and 333 in other mammalian GHRs, which were
considered to be necessary for the dimerization of GHR and the specific
GH-stimulated functions respectively, were replaced by tyrosine and serine in
gpGHR. Here, we report the functional expression of gpGHR and its mutants,
gpGHRY168H and gpGHRS332Y, in COS-7 cells and/or Chinese hamster ovary (CHO)
cells. It was shown that the COS-7 cells transfected with pcDNA3-gpGHR
possessed high affinity to bovine GH [K
molecular weight around 92 kD was detected by anti-mouse GHR monoclonal
antibody (mAb263). When CHO cells were transfected with the expression vectors,
pcDNA3-gpGHR, pcDNA3-gpGHRY168H and pcDNA3-gpGHRS332Y, the gpGHR and its
mutants were expressed and the ligand binding, phosphorylation of JAK2, protein
synthesis, and lipogenesis were studied. It was found that the mutation of
serine to tyrosine at position 332 greatly increased the GH-stimulated protein
synthesis and the phosphorylation of JAK2, while the mutation of tyrosine to
histidine at position 168 increased the protein synthesis and decreased the
phosphorylation of JAK2 only weakly. However, both mutations decreased the
GH-stimulated lipogenesis. Thus, our study provides the experimental evidence
that gpGHR may mediate the metabolic actions of GH and the substitutions of
some conserved amino acids in gpGHR result in the changes of post-binding
signaling.
Key
words guinea
pig; growth hormone receptor; mutagenesis; lipogenesis; protein synthesis
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