Ligand-Independent Homo- and Heterodimerization of Human Prolactin Receptor Variants: Inhibitory Action of the Short Forms by Heterodimerization

Abstract

Prolactin (PRL) acts through the long form (LF) of the human PRL receptor (hPRLR) to cause differentiation of mammary epithelial cells through activation of the Janus kinase-2 (JAK2)/signal transducer and activator of transcription 5 (STAT5) pathway and subsequent transcriptional events. To determine whether the inhibitory action of hPRLR short forms (SFs; S1a and S1b) on PRL-induced signal transduction through the LF results from heterodimerization, we studied complex formation among variant forms of the hPRLR. 3'-Tagged fusion constructs, with activities comparable to the wild-type species, were used to investigate homodimer and heterodimer formation. The LF and both SFs of the hPRLR formed homodimers under nonreducing conditions, independently of PRL, but formed only monomers under reducing conditions. Coimmunoprecipitation of the cotransfected LF with the SFs (S1a or S1b) in transfected cells showed ligand-independent heterodimerization of individual SFs with the LF. Bioluminescence resonance energy transfer analysis demonstrated homo- and heterodimeric associations of hPRLR variants in human embryonic kidney 293 cells. Biotin-avidin immunoprecipitation analysis revealed that hPRLR forms are cell surface receptors and that SFs do not influence the steady state or half-life of the LF. Significant homo- and heterodimerization of biotinylated membrane hPRLR forms was observed. These findings indicate that homo- and heterodimers of hPRLR are constitutively present, and that the bivalent hormone acts on the preformed LF homodimer to induce the active signal transduction configuration. Although SF homodimers and their heterodimers with LF mediate JAK2 activation, the SF heterodimer partner lacks cytoplasmic sequences essential for activation of the JAK2/signal transducer and activator of transcription 5 pathway. This prevents the heterodimeric LF from mediating activation of PRL-induced genes.

S179D prolactin: Antagonistic agony!

“…A potential clue here is offered by work from several other laboratories. First, it seems that growth hormone receptors, erythropoietin receptors and PRLRs are all dimerized prior to ligand binding (Brown et al, 2005;Lu et al, 2006;Qazi et al, 2006). This has long seemed more likely to the author since waiting for random collision of the second receptor with the complex between ligand and first receptor seemed inefficient.…”

The Role of Constitutively Active Prolactin Receptors in the Natural History of Breast Cancer

“…However, the level of endogenous full-length PRLR in the cells should also be considered. Several studies, including some from our lab, have noted heterodimerization of the long and short receptors can occur (18,19). In addition, Gadd et al report that artificially truncated PRLRs can also heterodimerize with endogenous receptors and alter PRL signaling (20).…”

Short form 1b human prolactin receptor down-regulates expression of the long form

“…In the mammary gland where PRL has also been suggested to play a role in disease, breast cancer progression is associated with a decreased ratio of short to long PRL receptors (Meng et al 2004). This progression has been attributed to loss of a dominant negative effect of the short receptors on signaling through the LF receptor, a dominant negative effect proposed to be produced by heterodimerization of short and long receptors (Trott et al 2003, Qazi et al 2006. Studies from this lab (Tan et al 2005) and the Dufau lab (Qazi et al 2006) have demonstrated that both homo-and hetero-dimers of long and short receptors are formed, and that ligands interact with both (Tan et al 2005).…”

“…This progression has been attributed to loss of a dominant negative effect of the short receptors on signaling through the LF receptor, a dominant negative effect proposed to be produced by heterodimerization of short and long receptors (Trott et al 2003, Qazi et al 2006. Studies from this lab (Tan et al 2005) and the Dufau lab (Qazi et al 2006) have demonstrated that both homo-and hetero-dimers of long and short receptors are formed, and that ligands interact with both (Tan et al 2005). The only pairing of these receptors that does not seem to occur is between the two different SFs (Tan et al 2005).…”

“…The traditional view has been that members of this cytokine family bring two (or more) receptors together, allowing transphosphorylation of the associated tyrosine kinases. It now seems that many (if not all) receptors are pre-dimerized and that ligands induce a change in conformation of the receptors which brings the tyrosine kinases together (Grotzinger 2002, Tan et al 2005, Walker 2005, Qazi et al 2006. Most of what we know about PRL receptor signaling comes from analysis of the LF, where the best studied pathway is activation of Jak2 and Stat5.…”