STOICHIOMETRIC STRUCTURE-FUNCTION ANALYSIS OF THE PROLACTIN RECEPTOR SIGNALING DOMAIN BY RECEPTOR CHIMERAS

The intracellular domain of the prolactin (PRL) receptor (PRLr) is req uired for PRL-induced signaling and proliferation, To identify and tes t the functional stoichiometry of those PRLr motifs required for trans duction and growth, chimeras consisting of the extracellular do main o f either the alpha or beta subunit of human granulocyte-macrophage col ony-stimulating factor (GM-CSF) receptor (GM-CSFr) and the intracellul ar domain of the rat PRLr were synthesized. Because the high-affinity binding of GM-CSF results from the specific pairing of one alpha- and one beta-GM-CSFr, use of GM-CSFr/PRLr chimera enabled targeted dimeriz ation of the PRLr intracellular domain, To that end, the extracellular domains of the alpha- and beta-GM-CSFr were conjugated to one of the following mutations: (i) PRLr C-terminal truncations, termed alpha 278 , alpha 294, alpha 300, alpha 322, or beta 322; (ii) PRLr tyrosine rep lacements, termed Y309F, Y382F, or Y309+382F; or, (iii) PRLr wild-type short, intermediate, or long isoforms, These chimeras were cotransfec ted into the cytokine-responsive Ba/F3 line, and their expression was confirmed by ligand binding and Northern and Western blot analyses, Da ta from these studies revealed that heterodimeric complexes of the wil d type with C-terminal truncation mutants of the PRLr intracellular do main were incapable of ligand-induced signaling or proliferation, Repl acement of any single tyrosine residue (Y309F or Y382F) in the dimeriz ed PRLr complex resulted in a moderate reduction of receptor-associate d Jak2 activation and proliferation, In contrast, trans replacement of these residues (i.e., alpha Y309F and beta Y382F) markedly reduced li gand-driven Jak2 activation and proliferation, while cis replacement o f both tyrosine residues in a single intracellular domain (i.e., alpha Y309+382F) produced an inactive signaling complex, Analysis of these GM-CSFr-PRLr complexes revealed equivalent levels of Jak2 in associati on with the mutant receptor chains, suggesting that the tyrosine resid ues at 309 and 382 do not contribute to Jak association, but instead t o its activation, Heterodimeric pairings of the intracellular domains from the known PRLr receptor isoforms (short-intermediate, short-long, and intermediate-long) also yielded inactive receptor complexes, Thes e data demonstrate that the tyrosine residues at 309 and 382, as well as additional residues within the C terminus of the dimerized PRLr com plex, contribute to PRL-driven signaling and proliferation. Furthermor e, these findings indicate a functional requirement for the pairing of Y309 and Y382 in trans within the dimerized receptor complex.