Characterization of the structure and glycosylation properties of intracellular and cell surface rat hepatic prolactin receptors.
In this study we examined the structure of the PRL receptor of rat liver. We used immunoblotting with monoclonal antibodies to binding and nonbinding site epitopes of the PRL receptor to assess receptor subtypes in different hepatic subcellular fractions. Analysis of frozen-thawed cell fractions revealed 40- and 42-kilodalton (kDa) species. Digestion with neuraminidase indicated that both species were terminally sialylated. Freshly isolated membranes exhibited a single 42-kDa species in all subcellular fractions, whereas freeze-thawing generated the 40-kDa species. The carbohydrate linkages present in the PRL receptor were examined using enzymes to deglycosylate iodinated purified receptors. These studies indicated that oligosaccharides comprise about 7 kDa of receptor mass and that they are exclusively N-linked, consisting of tri- and/or tetra-antennary complex glycans. Monoclonal antibodies to the receptor recognized the deglycosylated receptor. Maximally deglycosylated receptors retained about 85% of their binding capacity. After in vivo tunicamycin treatment of rats, total PRL receptors (as determined by immunoblot analysis and binding activity) disappeared with a half-time of about 25 min. In this circumstance, no aglycosylated receptor species were recognized by monoclonal antibodies. Since deglycosylation of mature receptors did not markedly reduce binding capacity, we infer that mature receptors do not accumulate during the blockade of glycosylation by tunicamycin. Thus, glycosylation appears to be required for the acquisition of a mature receptor status, but is not necessary for the maintenance of that status.
Dorato
A
A
Polypeptide Laboratory, McGill University, Montreal, Quebec, Canada.
Raguet
S
S
Okamura
H
H
Bergeron
J J
JJ
Kelly
P A
PA
Posner
B I
BI
D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D006031:Glycosylation; D015151:Immunoblotting; D007425:Intracellular Membranes / Q000378:metabolism; D008099:Liver / Q000378:metabolism*; D008862:Microsomes, Liver / Q000378:metabolism; D009439:Neuraminidase / Q000494:pharmacology; D011388:Prolactin / Q000378:metabolism; D051381:Rats; D011981:Receptors, Prolactin / Q000737:chemistry* / Q000187:drug effects / Q000378:metabolism; D012756:Sheep; D014357:Trypsin / Q000494:pharmacology; D014415:Tunicamycin / Q000494:pharmacology
D011981:Receptors, Prolactin; D014415:Tunicamycin; D011388:Prolactin; D009439:Neuraminidase; D014357:Trypsin