Expression of a functional porcine growth hormone receptor cDNA in mouse L cells.
Porcine (p) growth hormone receptor (GHR) complementary DNA (cDNA) has been cloned and the primary amino acid structure was deduced from the nucleotide sequence. A comparison of pGHR to other GHRs revealed an approximately 70% similarity in amino acid sequence (Cioffi et al., 1990). Hybridization of this receptor cDNA to RNA samples isolated from various porcine tissues revealed a single RNA band of 4.2 kb. The full-length pGHR cDNA was subcloned into an eukaryotic expression vector, transcription of which was directed by the mouse metallothionein-I transcriptional regulatory sequence. Stable mouse L cell lines which express the pGHR cDNA were established. Approximately 80% of the cell lines were found to possess pGHR mRNA (approximately 2 kb) which corresponds to the length of the cloned pGHR cDNA. Binding studies showed that the stable cell lines were capable of specifically binding 125I-labeled pGH with a dissociation constant (Kd) of approximately 1.0 nM. The apparent molecular mass of the receptor, as determined by cross-linking studies, was found to be 118 kDa. Also, the receptor-ligand complex could be internalized. These results suggest that an active form of pGHR had been cloned and stably expressed in mouse L cells.
Wang
X
X
Department of Biological Sciences, Ohio University, Athens 45701.
Cioffi
J A
JA
Kelder
B
B
Harding
P A
PA
Chen
W Y
WY
Kopchick
J J
JJ
D000595:Amino Acid Sequence; D000818:Animals; D001667:Binding, Competitive; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D005822:Genetic Vectors; D013006:Growth Hormone / Q000378:metabolism; D007739:L Cells / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D010957:Plasmids; D012333:RNA, Messenger / Q000235:genetics; D011986:Receptors, Somatotropin / Q000096:biosynthesis* / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics; D013552:Swine / Q000235:genetics
D012333:RNA, Messenger; D011986:Receptors, Somatotropin; D011993:Recombinant Fusion Proteins; D013006:Growth Hormone; D004247:DNA