N-glycosylation of the prolactin receptor is not required for activation of gene transcription but is crucial for its cell surface targeting.
The functional importance of the three oligosaccharide chains linked to Asn35, Asn80 and Asn108, of the long form of the PRL receptor (PRLR) was investigated by individual or multiple substitutions of asparagyl residues using site-directed mutagenesis and transient transfection of these mutated forms of PRLR in monkey kidney cells, Chinese hamster ovary, and human 293 fibroblast cells that exhibit different levels of protein expression. Scatchard analysis performed on monkey kidney cells revealed that the mutants possess the same affinity for PRL as compared with wild-type PRLR. A strong reduction (90%) of the aglycosylated PRLR expression at the cell surface of monkey kidney or human 293 cells was observed. Immunohistochemistry experiments using an anti-PRLR monoclonal antibody showed an accumulation of the deglycosylated receptor in the Golgi area of transfected monkey kidney cells. Upon PRL stimulation, the aglycosylated PRLR associated with Janus kinase 2 was phosphorylated and was able to activate a beta-casein gene promoter in transfected 293 fibroblast cells. The active form of the PRLR was thus acquired independently of glycosylation. By contrast, no functional activity was detectable in transfected Chinese hamster ovary cells that expressed low levels of PRLR. These studies demonstrate that the glycosylation on the asparagyl residues of the extracellular domain of the PRLR is crucial for its cell surface localization and may affect signal transduction, depending on the cell line.
Buteau
H
H
INSERM U344: Endocrinologie Moléculaire, Faculté de Médecine Necker, Paris, France.
Pezet
A
A
Ferrag
F
F
Perrot-Applanat
M
M
Kelly
P A
PA
Edery
M
M
D000818:Animals; D016466:CHO Cells; D019556:COS Cells; D002236:Carbohydrate Conformation; D002364:Caseins / Q000235:genetics; D002462:Cell Membrane / Q000378:metabolism; D006224:Cricetinae; D006031:Glycosylation; D006801:Humans; D007424:Intracellular Fluid / Q000378:metabolism; D053614:Janus Kinase 2; D008970:Molecular Weight; D011388:Prolactin / Q000494:pharmacology; D011401:Promoter Regions, Genetic / Q000187:drug effects; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D011518:Proto-Oncogene Proteins; D051381:Rats; D011981:Receptors, Prolactin / Q000235:genetics* / Q000378:metabolism* / Q000502:physiology; D015533:Transcriptional Activation / Q000502:physiology*; D014443:Tyrosine / Q000378:metabolism
D002364:Caseins; D011518:Proto-Oncogene Proteins; D011981:Receptors, Prolactin; D014443:Tyrosine; D011388:Prolactin; D011505:Protein-Tyrosine Kinases; C507924:JAK2 protein, human; C507927:Jak2 protein, rat; D053614:Janus Kinase 2