Modulation of growth factor receptor function by isoform heterodimerization.
Activation of prolactin (PRL)-dependent signaling occurs as the result of ligand-induced dimerization of receptor (PRLr). Although three PRLr isoforms (short, intermediate, and long) have been characterized and are variably coexpressed in PRL-responsive tissues, the functional effects of ligand-induced PRLr isoform heterodimerization have not been examined. To determine whether heterodimeric PRLr complexes were capable of ligand-induced signaling and cellular proliferation, chimeras consisting of the extracellular domain of either the alpha or beta subunit of human granulocyte-macrophage colony-stimulating factor receptor (GM-CSFr) and the intracellular domain of the rat intermediate or short PRLr isoforms (PRLr-I or PRLr-S) were synthesized. Because high affinity binding of GM-CSF is mediated by the extracellular domain of one alpha and beta GM-CSFr pair, use of GM-CSFr/PRLr chimera specifically directed the dimerization of the PRLr intracellular domains within ligand-receptor complexes. Stable transfection of these constructs into the Ba/F3 line was demonstrated by Northern blot and immunoprecipitation analyses. Flow cytometry revealed specific binding of a phycoerythrin-conjugated human GM-CSF to the transfectants, confirming cell surface expression of the chimeric receptors. When tested for their ability to proliferate in response to GM-CSF, only chimeric transfectants expressing GM-CSFr/PRLr-I homodimers demonstrated significant [3H]thymidine incorporation. GM-CSF stimulation of transfectants expressing either GM-CSFr/PRLr-S homodimers or GM-CSFr/PRLr-S+1 heterodimers failed to induce proliferation. Consistent with these data, the GM-CSF-induced activation of two phosphotyrosine kinases, Jak2 and Fyn, was observed only in homodimeric GM-CSFr/PRLr-I transfectants. These results show that the PRLr-S functions as a dominant negative isoform, down-regulating both signaling and proliferation mediated by the receptor complex. Thus, structural motifs necessary for Jak2 and Fyn activation within the carboxy terminus of the PRLr-I, absent in the PRLr-S, are required in each member of the dimeric PRLr complex.
Chang
W P
WP
Department of Pathology and Laboratory Medicine, University of Pennsylvania Medical Center, Philadelphia, 19104, USA.
Clevenger
C V
CV
D000818:Animals; D001704:Biopolymers; D002455:Cell Division; D002460:Cell Line; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000378:metabolism; D006801:Humans; D007536:Isomerism; D051379:Mice; D011388:Prolactin / Q000378:metabolism; D011485:Protein Binding; D051381:Rats; D016187:Receptors, Granulocyte-Macrophage Colony-Stimulating Factor / Q000378:metabolism*; D011981:Receptors, Prolactin / Q000737:chemistry* / Q000378:metabolism; D015398:Signal Transduction
D001704:Biopolymers; D016187:Receptors, Granulocyte-Macrophage Colony-Stimulating Factor; D011981:Receptors, Prolactin; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D011388:Prolactin