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Neuraminidase treatment of lactogen receptor-li2I-hGH complex CMF (lanes a-d) and AP (lanes e-h) preparations containing 750 jug of protein were incubated with 125I-hGH (5 x 105 c.p.m.) in the absence (-) or presence (+) of a 200-fold excess of unlabelled hGH at 20 °C for 18 h. Crosslinking and treatment of samples with neuraminidase was performed as described in the Materials and methods section. SDS/PAGE and autoradiography were carried out as described in the legend to Fig. 1.
Source publication
The types of carbohydrate chains present in a rat liver lactogenic hormone-binding receptor species with an Mr of 82,000, and in its hormone-binding subunits with Mr values of 40,000 and 35,000, were characterized using carbohydrate-chain-cleaving enzymes and affinity cross-linking. The subcellular distribution of lactogenic hormone-binding species...
Contexts in source publication
Context 1
... complex oligosaccharide chains often contain sialic acids (Sharon & Lis, 1982) we investigated whether this was the case with the complex chains of the Mr- 40000 and the major part of Mr-82000 entities. Fig. 3 shows the results of neuraminidase treatment of cross- linked M and AP preparations (Fig. 3, lanes a- Cross-linked RMF was shown to contain Mr-62000, -57000 and -54000 complexes (Fig. 4, lanes a and b) (Fig. 4, lanes c and d) or Triton X-100-solubilized (results not shown) preparations. Detergent-solubilized and cross-linked IMF, run ...
Context 2
... complex oligosaccharide chains often contain sialic acids (Sharon & Lis, 1982) we investigated whether this was the case with the complex chains of the Mr- 40000 and the major part of Mr-82000 entities. Fig. 3 shows the results of neuraminidase treatment of cross- linked M and AP preparations (Fig. 3, lanes a- Cross-linked RMF was shown to contain Mr-62000, -57000 and -54000 complexes (Fig. 4, lanes a and b) (Fig. 4, lanes c and d) or Triton X-100-solubilized (results not shown) preparations. Detergent-solubilized and cross-linked IMF, run on SDS/PAGE under non- reducing conditions, contained an additional Mr-104000 complex (results not ...
Citations
... Indeed, crosslinked complexes of 125 I-hGH with both long and short forms of PRL-R were immunoprecipitated by the S-46 polyclonal anti PRL-R antibody (Dusanter-Fourt et al., 1984), and were resolved by SDS-PAGE and autoradiography to the short form of the PRL-R have been isolated, which are identical up to aa 262 and diverge in the cytoplasmic domain (Davis and Linzer, 1989). At least part of this diversity was recently attributed to differences in the extent of glycosylation of the lactogenic receptor (Haldosen et al., 1989;Lascols et al., 1989;Dorato et al., 1992;Cahoreau et al., 1994;Lascols et al., 1994) or to ubiquitination (Cahoreau et al., 1994), causing alterations in electrophoretic mobility on SDS gels. While these differences could account, at least partially, for the divergence of the molecular weights measured, from the values calculated on the basis of sequence analysis, other post-translational modifications of the receptor cannot be ruled out. ...
The aim of this study is to further characterize the prolactin receptors (PRL-R) previously reported in the murine Leydig tumor MA-10 cell line, as well as to study their homologous and heterologous regulation. Two forms of PRL-R, a high and a low molecular weight form, were revealed by studies of covalent crosslinking of 125I-human GH to cultured MA-10 cells or cell membranes and immunoprecipitation of the solubilized PRL-R complexes with polyclonal anti PRL-R antibody, followed by SDS-PAGE and autoradiography. The long form had a molecular weight of 101 kDa and was predominant when the study was performed in the presence of protease inhibitors. The short form, with a molecular weight of 39 kDa, appeared, at least in part, to be a proteolytic product of the longer form. The same size forms of PRL-R were detected by crosslinking studies in the parental C57BL/6 mouse testicular Leydig cells, indicating the physiological relevance of the MA-10 cell model to the study of Leydig cell PRL-R. Homologous down-regulation of PRL-R was demonstrated in cultured MA-10 cells exposed for 24 h to increasing concentrations of PRL. In contrast, heterologous, 3–5-fold up-regulation of PRL-R was induced by various cAMP-elevating agents, including 8-bromo-cAMP (10−4–10−3 M), dibutyryl cAMP (3×10−3 M) and cholera toxin (1–10 ng/ml), although not by hCG (up to 100 ng/ml). This up-regulatory effect was apparently the result of a change in affinity, since cholera toxin caused a 2.4-fold increase in PRL-R affinity, with no change in the number of binding sites. In summary, these studies provide further evidence that MA-10 Leydig cells can serve as a physiologically relevant model for the study of PRL and PRL-R interactions, both at the functional level, as shown in our previous study, and at the structural and regulatory levels as shown in the current study.
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.
Basal parameters for binding and cross-linking of 125I-rat prolactin (rPRL) to lactogenic (PRL) binding species present in crude membrane fraction (CMF) or detergent-solubilized preparations of rat liver have been investigated. (1) The highest specific binding to CMF was obtained with an incubation time of 50 h at 20 degrees C and with a 50 mM potassium phosphate buffer adjusted to pH 8.5. (2) Cross-linking of 125I-rPRL to binding sites in CMF with disuccinimidyl suberate (DSS) showed the autoradiographic appearance of an Mr 40,000 binding species. (3) No specific binding or cross-linking of rPRL was seen in Triton X-100-solubilized CMF. This is probably due to Triton X-100-induced changes in the physical properties of rPRL. (4) Specific binding of 125I-rPRL was detected in CHAPS-solubilized CMF. Following cross-linking the autoradiographic appearance of a binding species with an Mr value of 40,000 was shown. 125I-hGH was cross-linked to three PRL binding species with Mr 82,000, 40,000 and 35,000 in CHAPS-solubilized preparations. (5) In Golgi-enriched low-density membrane preparation 125I-rPRL was cross-linked to Mr 82,000, 40,000 and 35,000 species. It is proposed that the inability of rPRL to be cross-linked to Mr 82,000 and 35,000 species present in CHAPS-solubilized preparation is the result of CHAPS-induced changes of rPRL binding properties and low solubilizing capacity of CHAPS. (6) In conclusion, this study shows that also the iodinated endogenous hormone, rat prolactin, and not only hGH identifies high and low molecular forms of the rat liver prolactin receptor.
