Growth hormone (GH) and prolactin (PRL) interact with GH and prolactin receptors (GHR; PRLR), causing activation of the JAK2 tyrosine kinase and the STAT5 and ERK pathways. We know much about how GH and PRL directly affect gene expression and cell behavior. Less is known about how they interact with other growth factors to modify signaling and biological responses when cells are exposed to GH or PRL and the growth factor -- a situation more like in vivo. This proposal addresses 2 aspects of """"""""crosstalk"""""""" between: 1) GH or PRL and epidermal growth factor (EGF) and 2) GH and insulin-like growth factor-1 (IGF-1). We recently reported that GH, via ERKs, causes threonine phosphorylation of EGF receptor (EGFR), retards EGF-induced EGFR downregulation, and enhances EGF-induced signaling in mouse preadipocytes. We observe similar crosstalk between PRL and EGF in T47D human breast cancer cells. We also find that GH promotes IGF-1 receptor (IGF-1R) association with the GHR-JAK2 complex and that IGF-1 enhances GH signaling. GH-IGF-1 crosstalk is not ERK-dependent, but may reflect IGF-1-induced changes in GHR activatability. We hypothesize: 1) GH and PRL, in addition to direct signaling, exert indirect effects to alter cellular EGF sensitivity by promoting EGFR threonine phosphorylation and influencing activated EGFR's intracellular itinerary. 2) GH collaborates with IGF-1 by using common signaling elements, broadening the idea that GH and IGF-1 exert independent and overlapping actions.
Our aims are: 1. Define mechanisms by which GH and PRL, via ERK activation, influence EGF signaling, EGFR trafficking, and biological effects. We will test GH effects on EGFR association/subcellular colocalization with proteins that mediate its downregulation. The impact of mutating an EGFR threonine phosphorylation site on EGFR downregulation and GH-EGF crosstalk will be analyzed in reconstitution systems. We will use bioluminescence imaging to study PRL-EGF crosstalk in T47D tumors in vivo. 2. Investigate the basis and significance of physical and functional interaction of GH and IGF-1 signaling elements. The role of JAK2 in GH-induced GHR-JAK2-IGF-1R complex formation will be determined and regions of GHR required will be mapped. GH-IGF-1 crosstalk will be studied in osteoblasts using a novel Cre-lox system to delete endogenous IGF-1R. These studies will significantly expand knowledge of GH and cytokine signaling and of biologically- relevant crosstalk between cytokine and growth factor signaling systems. ? ? ?
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