Characterization of signaling pathways involved in the homeostasis of the endosomal apparatus

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DESCRIPTION OF THE PROJECT: After insulin or EGF binding, tyrosine kinase-activated receptors (RTKs) internalize into the endosomal apparatus (EA). In endosomes, the signal can be amplified, selected, or terminated. Conversely, it is perceived that the EA controls the intracellular location and thus, the response of RTKs. This project is focused on the characterization of signaling mechanisms controlling the homeostasis of the EA. ORIGINALITY: During the last funding periods, we generated a library of hepatic endosomal proteins that are tyrosine phosphorylated in vivo. These proteins are being identified progressively and new mechanisms are characterized. We will focus here on the c-src/DPPIV pathway. OBJECTIVES: 1) To characterize DPP IV tyrosine-phosphosites as well as analyze the endosomal proteins associated with DPPIV and c-src. 2) To characterize the mechanism of action c-src in endosomes. 3) To pursue the proteomic characterization of endosomal fractions. SCIENTIFIC APPROACH: 1) Insulin, and presumably EGF, have the capacity to sort DPP IV to src-containing compartments. Here, DPPIV is tyrosine-phosphorylated. DPPIV phosphosites as well as the organization of the luminal c-src platform will be characterized in the different endosomal compartments. Fractionation in concert with biochemical approaches will be validated by confocal microscopy. 2) Highly purified hepatic endosomal fractions were characterized previously and we initiated a proteomic survey by identifying proteins present in a fraction comprised of Golgi/endosomes. We will pursue here, a systematic differential identification of proteins in early versus late vesicular compartments following insulin and EGF stimulation. SIGNIFICANCE; This program aims to clarify both the functional connections among elements of the endocytic pathway as well as elucidate mechanisms by which surface outputs are integrated.