Ligand binding to many membrane receptors initiates internalization of both ligand and receptor. This process has been shown to result in multiple potential fates, including recycling of receptors back to the surface, degradation of ligand and/or receptor by lysosomes or proteasomes resulting in down-regulation of displayed receptor, or modulation of signaling by temporally and spatially regulating the location of the receptor. Endocytosis is therefore a major modulator of cell responsiveness over the short term. We are only beginning to understand the molecular events regulating these processes for other classes of membrane receptors. Different receptor isoforms with distinct signaling capacities, such as those for the prolactin (PRL) receptor, are expressed on the same target cells, but at different levels depending on target and environment. Thus knowledge of these processes and net effect on PRL action is essential to understanding PRL function at its targets in physiologic and pathologic states. Our data demonstrate that PRL receptor isoforms are internalized at different rates, that ligand binding leads to down regulation of the """"""""long"""""""" but not the """"""""short"""""""" PRLR isoform, and that PRL-induced activation of c-Src is important for internalization of the """"""""long"""""""" but not the """"""""short"""""""" PRLR isoform. We hypothesize that ligand binding to the """"""""long"""""""" and """"""""short"""""""" PRLR isoforms results in receptor endocytosis and processing by distinct mechanisms dictated by sequences in the cytoplasmic domain, and that these events both are determined by transduced signals, and modulate net output. We propose to: 1. Compare endocytic pathways for each PRLR isoform using confocal microscopy, dominant negative constructs, and biochemical fractionation, and identify domains of the PRL receptors which are responsible. 2. Examine post-internalization sorting and fate of receptor isoforms in constitutive and ligand-induced endocytosis using selective inhibitors, biochemical and immunohistochemical approaches, and identify PRL receptor domains which participate. 3. Characterize the requirements for signal transduction in internalization and trafficking of the isoforms, and determine the relationship of endocytosis to signal transduction through several pathways. 4. Determine the role of dimerization in this process, and the effect of homo- vs. hetero-dimerization using chimeric receptors and PRL antagonists.
