DOMINANT-NEGATIVE AND COOPERATIVE EFFECTS OF MUTANT FORMS OF PROLACTIN RECEPTOR

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 dom ains, have been identified in the rat. The Nb2 form, lacking 198 aa in the cytoplasmic domain, is able to transmit a lactogenic signal simil ar 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 th e lactogenic hormone responsive element fused to the luciferase report er was assessed in Chinese hamster ovary cells or 293 fibroblasts tran siently transfected with PRLR cDNAs. The function of the short form wa s examined after cotransfection of both the long and short forms. Thes e results clearly show that the short form acts as a dominant negative inhibitor through the formation of inactive heterodimers, resulting i n 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 cyt oplasmic receptors in the process of signal transduction. We cotransfe cted Chinese hamster ovary cells with two cDNA constructs: a cytoplasm ic (soluble) form of the receptor with a deleted signal peptide (Delta -19), which is unable to bind PRL, and a functionally inactive recepto r 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 efficie ncy), providing that association/activation occurs with a PRL-PRLR com plex initiated at the cell surface level; box 1 of the cytoplasmic for m 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 i mplicated JAK2. Our results provide evidence that heterodimerization o f receptors can be implicated either in the positive or in negative ac tivation of gene transcription.