| Abstract: |
Incubation of hepatocytes from pregnant rats with dithiothreitol decreased specific 125I-prolactin (125I-prl) binding to such cells by about 20% relative to control. This was not due to a non-specific effect of dithiothreitol on the cell membrane, since reduction also altered the binding of prl to solubilized partially purified receptor. Exposure of hepatocytes to N-ethylmaleimide (6 mM) for periods as brief as 1 min decreased the subsequent specific binding of 125I-prl by more than 50%. N-Ethylmaleimide was less effective as an inhibitor of binding when applied after hepatocytes had been exposed to 125I-prl, binding being decreased by about 15%. Scatchard analysis demonstrated that the effect of N-ethylmaleimide resulted from loss of receptor-binding capacity without any substantial effect on the affinity of the prl receptor for hormone. Dithiothreitol diminished the affinity of lactogenic sites for prolactin without altering cellular binding capacity. These observations suggest that thiol and disulphide groups are present in the prl receptor and that these functional moieties regulate the formation and properties of prl receptor complexes. The species to which 125I-prl had bound were identified by affinity labelling. 125I-prl was covalently coupled into saturable complexes of M(r) 65,000 and 50,000. 125I-human growth hormone (125I-hGH) was covalently incorporated into complexes of M(r) 300,000, 220,000, 130,000, 65,000 and 50,000. Bone growth hormone (bGH), but not prl, competed for 125I-hGH uptake into the 300,000-, 220,000- and 130,000-M(r) complexes, indicating that these species were somatogenic. Prl, but not bGH, inhibited 125I-hGH uptake into 65,000- and 50,000-M(r) complexes. This demonstrated that 125I-hGH in the presence of bGH could affinity-label lactogenic receptors. 125I-prl aggregates in Triton X-100, whereas 125I-hGH does not. Therefore lactogenic complexes to which 125I-hGH was bound in the presence of excess bGH were solubilized in Triton X-100 and characterized sequentially by gel filtration and affinity labelling. Prl receptors were eluted from columns of Sepharose 6B as a species of M(r) 380,000. Fractionation of the 380,000-M(r) species on sodium dodecyl sulphate polyacrylamide gels resulted in the isolation of complexes of M(r) 65,000 and 50,000. Thus non-covalent forces stabilize aggregates of the monomeric prolactin receptor. |
