Endocytosis and degradation of prolactin and its receptor in Chinese hamster ovary cells stably transfected with prolactin receptor cDNA.
Molecular cloning of the prolactin (PRL) receptor cDNA has revealed different forms of the receptor: among them, the longest form encodes a transmembrane protein of 592-598 amino acids and was originally found in rabbit mammary gland as well as in human and rat tissues. It contains a cytoplasmic domain of 358 amino acids. In CHO cells transfected with the PRL receptor cDNA, PRL is able to induce the specific expression of a reporter gene provided with the promoter of the milk protein gene beta-lactoglobulin. The cDNA encoding this long receptor form has been expressed permanently after stable transfection of Chinese hamster ovary (CHO) cells. In these cells, we have determined the fate of the bound hormone and of the receptor. At 37 degrees C, transfected cells were able to endocytose 125I-labeled human growth hormone (hGH) or ovine prolactin (oPRL) at an initial rate of about 1 fmol/h at 100 pM labeled hormone and 10(6) cells/well. Lowering the temperature to 15 degrees C slowed the endocytosis of [125I]hGH by a factor of 5. These results were confirmed by electron microscopy with oPRL labeled with colloidal gold. At 37 degrees C, the receptor underwent rapid insertion to the cell surface and constitutive endocytosis (half-life 80 min). This rate of endocytosis was enhanced in the presence of 10 nM oPRL (half-life 8 min), leading to down-regulation of the receptor by exhaustion of the intracellular receptor pool. After down-regulation, the cell surface was replenished with newly synthesized PRL receptor with a half-time of 8-10 min. If cycloheximide was added, almost no receptors could be found on the cell surface. These results indicate that in transfected cells the PRL receptor behaved largely as in classical target cells. A "conveyor belt" endocytosis behavior was found, with degradation of the endocytosed receptors, and occupation by the hormone enhancing this process. Moreover, since the PRL receptor belongs to a family of receptors in which companion protein(s) seem to play important roles, transfected CHO cells appear to provide the expressed receptors with the necessary element(s) to function as in normal PRL target cells.
Genty
N
N
Unité d'Ingéniérie des Protéines, INRA-Biotechnologies, Jouy-en-Josas, France.
Paly
J
J
Edery
M
M
Kelly
P A
PA
Djiane
J
J
Salesse
R
R
D000818:Animals; D001667:Binding, Competitive; D016466:CHO Cells; D003001:Cloning, Molecular; D006224:Cricetinae; D003513:Cycloheximide / Q000494:pharmacology; D018076:DNA, Complementary / Q000235:genetics*; D004705:Endocytosis; D005455:Fluorescent Antibody Technique; D013006:Growth Hormone / Q000378:metabolism; D007700:Kinetics; D008854:Microscopy, Electron; D011388:Prolactin / Q000378:metabolism*; D011981:Receptors, Prolactin / Q000235:genetics / Q000378:metabolism*; D014162:Transfection
D018076:DNA, Complementary; D011981:Receptors, Prolactin; D011388:Prolactin; D013006:Growth Hormone; D003513:Cycloheximide