Dominant Negative and Cooperative Effects of Mutant Forms of Prolactin Receptor

Abstract

In addition to a long form of 591 amino acids (aa), two other forms of PRL receptor (PRLR), differing in the length of their cytoplasmic domains, have been identified in the rat. The Nb2 form, lacking 198 aa in the cytoplasmic domain, is able to transmit a lactogenic signal similar to the long form, whereas the short form of 291 aa is inactive. The ability of PRL to activate the promoter of the beta-casein gene or the lactogenic hormone responsive element fused to the luciferase reporter was assessed in Chinese hamster ovary cells or 293 fibroblasts transiently transfected with PRLR cDNAs. The function of the short form was examined after cotransfection of both the long and short forms. These results clearly show that the short form acts as a dominant negative inhibitor through the formation of inactive heterodimers, resulting in an inhibition of Janus kinase 2 (JAK2) activation. The present study also investigates the possible participation of cytoplasmic receptors in the signal transduction pathway, using cotransfection experiments and a new approach that selectively determines the contribution of cytoplasmic receptors in the process of signal transduction. We cotransfected Chinese hamster ovary cells with two cDNA constructs: a cytoplasmic (soluble) form of the receptor with a deleted signal peptide (delta-19), which is unable to bind PRL, and a functionally inactive receptor mutant (lacking box 1), which is anchored in the plasma membrane and able to bind PRL. This approach has allowed us to show that delta-19, lacking expression at the plasma membrane, can transduce the hormonal message, at least to a limited extent (up to 30% of wild type efficiency), providing that association/activation occurs with a PRL-PRLR complex initiated at the cell surface level; box 1 of the cytoplasmic form is necessary to rescue this partial transcriptional activity of the inactive mutant. This partial recovery is also parallel to the partial activation of JAK2, indicating that the signal transduction pathway implicated JAK2. Our results provide evidence that heterodimerization of receptors can be implicated either in the positive or in negative activation of gene transcription.

Quantitation of prolactin receptor mRNA in the maternal rat brain during pregnancy and lactation

“…It is possible that an increasing ratio of short-to long-form PRL-R may contribute to the impaired prolactin signalling in these neurones at this time. The short form of the receptor may act as a competitive inhibitor by forming inactive heterodimer receptor complexes with the long form (Perrot-Applanat et al 1997). Such an effect might contribute to the loss of sensitivity of TIDA neurones to prolactin during late pregnancy and lactation.…”

Long isoform of prolactin receptor predominates in rat intrahepatic bile ducts and further increases under obstructive cholestasis

“…The long PrlR isoform activates various signalling cascades, among which Janus kinase/signal transduction and activators of transcription (JAK/STAT) is considered to be the central pathway (Bole-Feysot et al 1998). The short PrlR isoform exerts dominant-negative effects on STAT activation, resulting from formation of short and long PrlR heterodimers (Perrot-Applanat et al 1997). However, short PrlR is also involved in transduction of specific signals, including activation of serine/threonine protein kinase cascades and interaction with 17-hydroxysteroid dehydrogenase (Das & Vonderhaar 1995, Duan et al 1997, Nikelainen et al 1998.…”

Cloning and expression of a unique short form of the porcine prolactin receptor

“…The pPRLR-SF functions as a dominant negative with an effectiveness that is very similar to the rat PRLR-SF (Perrot-Applanat et al 1997). The pPRLR-SF does not appear to affect the level of pPRLR-LF protein and co-transfection of pPRLR-SF and pPRLR-LF increases the level of specific PRL binding.…”