Duncan I. Mackie - US grants

Abstract G-protein coupled receptors (GPCRs) represent the largest class of cellular receptors. Signaling by GPCRs involves activation by ligands, resulting in signal transduction to the interior of the cell through changes in their structure. These receptors mediate physiological processes such as neurotransmission, cellular differentiation, growth, and immune responses. Because of this control GPCRs have emerged as major targets for the drug discovery. It is estimated that ~60% of all clinically prescribed xenobiotics target GPCRs. With this successful targeting, it has become widely acknowledged that their regulation is very complex. Additionally, there are several known families of GPCR interacting proteins. The most studied example of GPCR-interacting partners that regulate receptor pharmacology is the receptor activity modifying proteins (RAMP1, -2 and -3). These proteins were identified in experiments focused on the calcitonin gene-related peptide (CGRP). It was determined that the functional CGRP receptor required the expression of the calcitonin receptor-like receptor (CLR) with the protein partner RAMP1. Subsequently, when CLR was co-expressed with RAMP2, this resulted in the formation of receptors activated not by CGRP, but rather by a related peptide known as adrenomedullin (AM). Since these early studies nine GPCRs have been shown to interact with the RAMP family members. The Caron lab has a long standing interest in RAMP: GPCR interactions and their functional consequences on lymphangiogenesis, cardiac development, and their involvement during pregnancy. Previous studies in our lab have shown that a certain receptor serves as a ?decoy? receptor for AM which consequently provides fine- tuned control of AM-mediated proliferation of LECs. Based on this decoy activity, expression patterns, and my preliminary studies, I propose to investigate the hypothesis that this ?decoy? receptor also modulates the pro- angiogenic sprouting of new blood vessels and that this activity is further regulated by novel GPCR: RAMP interactions. I will utilize in vitro approaches to confirm the interaction between two new GPCR: RAMP partners. Furthermore, I will use KO mice we currently possess in the metatarsal and matrigel plug assays to examine the effects of the genetic depletion of these GPCRs and RAMPs on angiogenesis. Finally, I will adapt a unique screening paradigm to interrogate the ?druggable? GPCR-ome in a high-throughput platform to elucidate previously unknown GPCR: RAMP interactions. This is essential for developing therapeutics that might target the various RAMP-interacting receptors. Results from this proposal will confirm my preliminary data of the existence of at least two new GPCR: RAMP interactions (Aim 1). It will determine, ex vivo and in vivo, whether the ?decoy? activities and/or RAMPs influence angiogenesis (Aim 2). Lastly, it will provide unique insight into previously uncharacterized G protein signaling modifications and provide new drug targets (Aim 3). This will allow for the development of a research program upon which I can eventually build my own research.