Expression of functional growth hormone receptor in a mouse L cell line infected with recombinant vaccinia virus.
The growth hormone receptor is a member of a large family of receptors including the receptors for prolactin and interleukins. Upon binding to one molecule of growth hormone two growth hormone receptor polypeptides dimerize. We have expressed the rabbit growth hormone receptor DNA in transfected mouse L cells infected with polymerase T7-producing vaccinia virus. The growth hormone receptor was synthesized as a 85-kDa protein and transported to the cell surface. Western blotting and metabolic labeling combined with immunoprecipitation using a rabbit antibody probably directed against the cytosolic domain of the receptor showed that its expression was dependent on both transfection of the growth hormone receptor DNA and vaccinia infection. Binding studies with 125I-labeled growth hormone demonstrated specific binding sites at the cell surface 20 h after transfection. Permeabilization with saponin showed that the growth hormone receptor binding sites were almost exclusively present at the cell surface with little intracellularly. Chemical crosslinking of the 125I-growth hormone complex resulted in a 180-kDa complex which could specifically be immunoprecipitated with the antiserum. Electron microscopic immunocytochemistry confirmed the presence of growth hormone receptor at the cell surface. Furthermore, specific growth hormone receptor antigen was also associated with intracellular membranes. These results thus show that this transient transfection system will be useful for cell biological studies of growth hormone receptor regulation.
Strous
G J
GJ
Department of Endocrinology, Medical School, Utrecht University, The Netherlands.
van Kerkhof
P
P
Verheijen
C
C
Rossen
J W
JW
Liou
W
W
Slot
J W
JW
Roelen
C A
CA
Schwartz
A L
AL
D000818:Animals; D000941:Antigens / Q000235:genetics; D003600:Cytosol / Q000378:metabolism; D004274:DNA, Recombinant / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D013006:Growth Hormone / Q000378:metabolism; D007739:L Cells; D051379:Mice; D016253:Microscopy, Immunoelectron; D011817:Rabbits; D011986:Receptors, Somatotropin / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D011995:Recombination, Genetic; D014162:Transfection; D014616:Vaccinia virus / Q000235:genetics
D000941:Antigens; D004274:DNA, Recombinant; D011986:Receptors, Somatotropin; D013006:Growth Hormone