Intramolecular disulfide bonds of the prolactin receptor short form are required for its inhibitory action on the function of the long form of the receptor.

The prolactin receptor (PRLR) belongs to the class I cytokine receptor superfamily (4, 5). It binds the pituitary hormone prolactin (PRL) with high affinity and triggers intracellular responses that participate in diverse biological functions in target tissues, including the mammary gland, organs of the reproductive system, the central nervous system, pituitary, and adrenal. At least nine variants are generated by alternative splicing of the human PRLR (hPRLR) gene (16-18, 21, 35); they differ in the lengths and compositions of their cytoplasmic domains and/or extracellular (EC) domains (http://atlasgeneticsoncology.org/Genes/PRLRID42891ch5p14.html). The full-length receptor, or long form (LF), is composed of a ligand binding EC domain, a single transmembrane domain, and a cytoplasmic domain required for signal transduction (5, 20). PRL acts through the LF to stimulate cell proliferation and differentiation. The short forms (SFs) of the receptor, S1a and S1b, are derived from alternative splicing of exons 10 and 11 and contain unique cytoplasmic sequences (16). S1a is a 376-amino-acid (aa) receptor that contains partial exon 10 sequences and a unique 39-aa C terminus derived from exon 11. S1b is a 288-aa variant that lacks the entire exon 10 and contains 3 aa derived from exon 11 at the C terminus. Unlike the LF, neither of the SF receptors can mediate activation of the β-casein gene promoter induced by PRL, and both SFs are inhibitory of the transcriptional activation induced by PRL through the LF (16). S1b is more effective than S1a in inhibiting LF actions due to its higher stability (16). The reduced expression of S1a and S1b relative to the LF in breast cancer tissues and cell lines, compared to adjacent normal tissues/cells, suggests that the relatively reduced expression of SFs in cancer could lead to gradations of unopposed PRL-induced LF stimulatory function and contribute to breast tumor development and progression (28). The initiation of signal transduction associated with members of the cytokine receptor family depends on the interaction of cognate ligand preformed receptor dimers (9, 13, 24, 32). In the case of the PRLR, immunoprecipitation studies (12, 31) and bioluminescence resonance energy transfer using coelenterazine (BRET1) analysis (31) demonstrated that hetero- and homodimerization of hPRLR can occur independently of ligand binding. This indicated that PRL is a conformational modifier that induces activation of the JAK2/STAT5 pathway through the LF and possibly through other JAK2-dependent pathways via the SFs. The dominant-negative effect of the SFs results from their heterodimerization with the LF, with the consequent functional inactivation due to the absence of cytoplasmic sequences of the dimerized LF partner (SF) required for downstream JAK2/STAT signaling (16, 31). Compared to the LF, S1b does not contain the conserved cytoplasmic structural motif beyond the Box-1 JAK2 docking site. In the homodimerized LF, the ligand-induced activation of JAK2 phosphorylates the receptor, preferentially at Y580 (rat) or Y587 (human) in its C terminus (2, 22). This induces phosphorylation, dimerization, and nuclear translocation of STAT5, which causes transcriptional activation of PRL-responsive genes, including the β-casein gene (8). Alternatively, Src has been demonstrated to participate in PRL-induced LF-mediated biological function by recruiting either Fak, Erk1/2, or P13K independently of the classic JAK2/STAT5 pathway (1, 5). These additional pathways have been proposed to operate in SF-mediated action in rodent species (10). Our studies of the mutagenesis of unpaired conserved cysteines adjacent to and within the transmembrane region of the PRLR (S1b) showed no significant changes in receptor homo- or heterodimerization (31). In the present study, we evaluated the relevance of the four conserved extracellular Cys residues that form two intramolecular disulfide (S-S) bonds in subdomain 1 (D1) of the EC domain to the formation of homo- and/or heterodimers and their relevance to the inhibitory function of the SF on the LF. These residues are known to be crucial for ligand binding (33) and hence for PRL-induced activation of the JAK2/STAT5 pathway through the PRLR LF and also presumably in the hormonal activation of S1a and S1b. These intramolecular S-S bonds might provide strong spatial constraints for tertiary folding. Moreover, these bonds might control the quaternary arrangement of the receptor, affecting the formation of homo- and/or heterodimers and the inhibitory function of the SF on the LF. We observed that these residues are essential for maintaining the conformation that favors SF/LF heterodimerization and are also important for the constitutive activity of JAK2 bound to the SF. These studies further revealed that the intramolecular S-S bonds are relevant to the inhibitory action of the SF on the LF function. In addition, molecular modeling of the PRLR wild type and Cys mutants in the EC domain provides insights into the molecular mechanism by which the structure impacts homodimer/heterodimer formation, JAK2 association, and the PRL-induced PRLR action.