MUTATIONAL ANALYSIS OF THE LIGAND-BINDING DOMAIN OF THE PROLACTIN RECEPTOR

The recent isolation and sequencing of the rat liver prolactin (PRL) receptor cDNA (clone F3) revealed that the receptor is a small molecular weight protein (nonglycosylated form, M(r) 33,000; glycosylated form, M(r) 42,000) comprised of 291 amino acids. A second form of the PRL receptor exists (591 amino acids) that contains a much longer cytoplasmic domain. In the present study, site-directed point mutations of the 5 conserved cysteine (Cys) residues and of the three potential N-linked glycosylation sites in the extracellular domain of the rat PRL receptor were constructed to assess their involvement in hormone binding. In addition, a truncation mutant (T-DELTA-237) lacking 55 of 57 intracellular amino acids was constructed to determine the influence of the cytoplasmic domain on ligand-receptor interactions. Binding studies of transiently transfected COS-7 cells demonstrated that serine substitution of the first 4 Cys residues (Cys12, Cys22, Cys51, and Cys62) completely eliminated binding of I-125-ovine PRL and I-125-U5 and -U6, two monoclonal antibodies that bind the receptor molecule outside the PRL-binding domain. RNA blot analysis of the transfected cells showed that both the wild-type and mutant clones had similar levels of expression of receptor mRNA. Immunoblot analysis demonstrated that lack of PRL binding in these mutants was not due to incomplete processing of the protein, since the fully glycosylated M(r) 42,000 form of the receptor was seen. Mutation of Cys184 had no effect on affinity or dimerization capacity of the receptor, suggesting the 5th cysteine is not directly involved in the binding domain. Carbohydrate groups of some receptors have been shown to be involved in ligand-receptor interactions as well as intracellular trafficking. This does not appear to be the case for the PRL receptor, since there was no corresponding decrease in affinity for PRL or cell surface receptor expression, following mutation of each of the 3 asparagine residues to aspartate. Interestingly, T-DELTA-237 showed a 4-5-fold increase in affinity for PRL as well as a marked increase in the number of receptor sites. Whole cell binding assays also demonstrated that loss of the cytoplasmic domain lead to inefficient recycling of the receptor. These studies suggest that the first 4 conserved Cys residues are crucial for ligand binding. The putative pattern of disulfide bridging between the 1st and 2nd and between the 3rd and 4th cysteines (Fuh, G., Mulkerrin, M. G., Bass, S., McFarland, N., Brochier, M., Bourell, J. H., Light, D. R., and Wells, J. A. (1990) J. Biol. Chem. 265, 3111-3115) provides strong spatial constraints for tertiary folding of the receptor. Disruption of these bonds leads to the formation of abnormally folded receptors and thus to the loss of the hormone-binding site. The studies also suggest that the cytoplasmic domain not only influences binding but also regulates the cellular distribution of the PRL receptor.