Immunological relationship and binding capacity of prolactin receptors in cytosolic and membrane fractions of rabbit mammary gland

Abstract

We have recently identified and partially characterized a specific lactogen binding protein in rabbit mammary gland cytosol. In this report, studies using pregnant or lactating rabbits are described which further characterize the cytosolic lactogen binding protein in relation to the membrane-bound lactogen receptor. The data show that in pregnant or lactating rabbits the binding capacity (fmol/mg protein) of membranes is at least double that of the cytosol preparation although when expressed on a tissue content basis (fmol/g tissue) there was no membrane-cytosol difference in receptor number. Treatment of lactating rabbits with CB-154, however, caused a marked increase (100–150%) in the binding capacity of membrane-bound receptors with comparatively little effect (+20%) on the cytosolic lactogram binding protein. There was also a marked difference in the association constants for 125I-hGH, with the cytosolic lactogen binding protein exhibiting a 6-fold higher affinity than the membrane-bound receptor. Three anti-prolactin receptor monoclonal antibodies (M110, A82 (antagonists) and A917 (agonist)) have also been used to assess the relative immunological characteristics of the cytosolic lactogen binding protein and the membrane lactogen receptor. Each monoclonal antibody was able to inhibit the specific binding of 125I-hGH to both membranes and cytosol in a dose-dependent manner. However, the order of potency was not identical being M110 > A917 > A82 in membranes and M110 > A82 > A917 in cytosol. A917 was at least 10 times more active in membranes than cytosol whereas A82 was at least 10 times more active in cytosol. Thus the cytosolic lactogen binding protein shares common but not identical immunological epitopes with the classical membrane receptor. The data presented here indicate that the cytosolic lactogen binding protein can be differentiated from the membrane-bound receptor in terms of its affinity for hGH and interaction with specific monoclonal antibodies. Further, the studies with CB-154 indicate that the cytosolic lactogen binding protein may be under different physiological control.