In this study, we have provided evidence, derived from functional analysis, that the four conserved Cys residues in the D1 subdomain of the EC region of the hPRLR are essential to preserve the dominant-negative action of the S1b SF on ligand-induced LF-mediated STAT5-dependent transcriptional activation. These conclusions were supported by our computer modeling, which provided insights into the effects of the S-S bonds on the stabilization of the tertiary and quaternary structure of the receptor. Our studies have demonstrated that the inhibitory action of wild-type S1b on the LF function was abolished upon disruption of these S-S bonds. The lack of inhibitory action of mutated S1b forms may result from their higher affinity for forming homodimer association than the wild type (Fig. ) and the significant reduction in their affinity for forming heterodimers with the LF (Fig. ). The observed reduction in the affinity (Fig. ) between LF/S1b4x and LF/S1b was consistent with a marked reduction in heterodimer formation revealed by co-IP studies (Fig. , lane 6 versus lane 4). Moreover, the marked increase in the affinity of the SF mutant (S1b4x-RL/S1b4x-Y) versus the wild type (S1b-RL/Sb-Y) was also reflected in the co-IP studies (Fig. , lane 8 versus lane 10). The preference of the mutated S1b to form homodimers rather than heterodimers contrasted with the wild-type S1b propensity to form heterodimers with the LF. This in turn facilitated the formation of LF homodimers competent to mediate PRL-induced downstream signaling. Thus, the EC Cys mutant lacks the inhibitory action normally observed in the wild-type SF. Also, these studies demonstrated that either of the Cys pairs (36/46 and 75/86) was important in the heterodimerization process and for the inhibitory effect of the SF on LF-mediated PRL action.
It is not surprising that S1b mediated JAK2 phosphorylation activity upon PRL stimulation, since it contains a conserved proline-rich domain at the Box-1 docking site for JAK2 association that is known to engage constitutively with the PRLR LF (22
). Janus kinases can trans
phosphorylate themselves and/or undergo autophosphorylation (36
). It is believed that the ligand, as the inducer of receptor aggregation or through conformational changes of existing dimers, causes tyrosine phosphorylation of the kinase at the activation loop and increases its catalytic activity. The significant increases in JAK2 phosphorylation induced by PRL in cells stably expressing the LF transfected with S1b (S1b versus control) reflects the additive effect of S1b and LF over the basal level present only in S1b (Fig. ). JAK2 activation associated with the SF with a short cytoplasmic sequence could have an impact on other potential signaling via mitogen-activated protein kinase, AKT, or JUNK, in contrast to STAT activation resulting from coupling to the additional cytoplasmic tail found in the LF. It was interesting to observe basal levels of JAK2 phosphorylation with S1b in the absence of PRL stimulation in cells both transiently and stably expressing S1b (Fig. ). Since no detectable basal JAK phosphorylation was associated with LF, it is possible that an impact of its extended cytoplasmic sequence on the overall conformation of the Box-1 region in the LF could prevent productive basal JAK2 phosphorylation. Thus, in the absence of hormone it appears that the shortened length of the cytoplasmic domain in S1b with only a breve sequence beyond Box-1 (8 aa identical to the LF and 3 unique aa) (16
) would favor basal JAK2 phosphorylation (at Tyr 1007/1008 in the activation loop), presumably via auto- and/or trans
phosphorylation. It is conceivable that the S1b association with JAK2 induces partial relaxation of the inhibition of the catalytic domain (JH1) by the pseudokinase (JH2) to cause basal activation. Additional disruption of the inhibition upon activation by a hormone would result in a further enhanced level (over the basal level) of JAK2 catalytic activity (Fig. ) (19
). This could also apply to heterodimers, since basal JAK2 phosphorylation is not present with LF homodimers in the absence of hormone (Fig. , control). Also, this may involve other associated kinase modules, as is the case for Src kinases in the activation of focal adhesion kinases (7
). In contrast to S1b, complete loss of basal JAK2 phosphorylation of its Cys mutant (S1b4x-Y) was observed in transiently transfected cells. The apparent loss of JAK2 binding to S1b4x compared to the S1b wild type observed in the co-IP study revealed (Fig. , right) a functional link between the EC domain conformation and the JAK2 association with its docking site at Box-1.
Wild-type S1b and the Cys mutant S1b4x were found to be similarly expressed at the cell surface by a biotin-avidin labeling procedure (Fig. ). This accounted for the effect of S1b on LF action at the cell membrane level and further validated the relevance of intramolecular Cys-Cys residues in the dominant-negative function of S1b on LF action. The loss of hormone activation of JAK2 in the Cys mutant of the S1b homodimer was expected, since these residues were found to be required for hormone binding by the LF homodimer in a point mutation study (33
). In addition, impairment of JAK2 association with the Cys-mutated receptor demonstrated in this study (Fig. ) would render the receptor unresponsive to the hormonal stimulus. Thus, it is probable that the spatial constraints conveyed by the S-S bonds on tertiary folding of the PRLR, which is essential for PRL binding, would also participate in the association/dissociation properties of dimer/ heterodimer variants and/or their productive association with JAK2.
The molecular dynamics simulations of the EC domain of the wild-type and of the Cys mutant monomers showed substantial conformational changes in the latter, with less bending of D1 with respect to D2. These changes are triggered by the structural relaxation of the native-like D1 subdomain (Fig. ). In contrast, the wild-type monomer remained practically unchanged throughout the dynamics (Fig. ). The simulations were based on the X-ray structure of the EC domain of hPRLR and rPRLR (PDB codes 1bp3 [monomer] and 1f6f [dimer template]). The decrease in the angle between D1 and D2 observed during the simulations implies that both domains tend to align in the mutant monomer (ω, ~30° to 40°), in contrast to the wild type, for which the relative orientation of the domains remained stable (ω, ~70° to 90°) (Fig. ). The surface area buried in the hormone-receptor interface resides mainly in the subdomain D1 of the receptor, as suggested by the X-ray structure of the hGH-hPRLR complex (34
). The conformational changes observed in the mutant reduce the accessible space for a ligand to bind the dimerized receptor. The simulations also showed that additional intermolecular H bonds can form in the mutant homodimers: in addition to the H bonds linking both D2 subdomains observed in the wild-type homodimer, several other residues form H bonds between the D1 subdomains. These additional H bonds in the mutant receptor are responsible for stabilizing the locked form of the dimer and may serve two functional roles: (i) prevent ligand binding to the receptor and (ii) increase the homodimer affinity of S1b4x. Estimation of the electrostatic potential on the molecular surface shows a positive region in the wild-type dimer interface (Fig. , top) surrounded by a predominately negative potential. This electrostatic pattern could be a motif for ligand recognition and binding, which is absent in the mutant (Fig. , top). The closure of the mutant binding site also explains structurally, based on simple steric arguments, why PRL may not bind to the PRLR mutant (33
). Several of the H-bonded residues located in the D2 domains of the X-ray structure have been implicated in the formation of the receptor-receptor interface of dimerization (34
). It is possible, then, that the additional H bonds observed in the mutant dimers could enhance the dimerization process and explain the comparatively significantly lower BRET50
Overall, the study reported here demonstrated the relevance of the S-S bonds of the PRLR for S1b inhibitory action on PRL-induced LF-mediated STAT5-dependent action and for cytoplasmic events related to JAK2 association/activity.