This proposal examines the general hypothesis that the neuropeptides substance P (SP) and calcitonin-gene related peptide (CGRP) act in a co-operative fashion to induce neurogenic inflammation of the intestine. The previous grant defined the mechanisms by which SP interacts with the neurokinin 1 receptor (NK1R) to initiate inflammation. Degradation of SP by the cell surface-enzyme neutral endopeptidase (NEP) and desensitization of the NK 1 R by Beta-arrestins, were found to terminate signaling by SP. In the current proposal, a combination of studies in transfected cell lines, endothelial cells that naturally express the receptors, and knockout mice that lack the peptides, their receptors, NEP and p-arrestins will be used to define novel mechanisms of neurogenic inflammation.
Aim 1 will define whether a complex of calcitonin receptor like receptor (CRLR) and receptor activity modifying protein (RAMP) 1 form a functional CGRP receptor that mediates the proinflammatory effects of CGRP. The mechanism and function of trafficking of CRLR/RAMP1 to and from the plasma membrane will be investigated by expression of dominant negative mutants of dynamin and rab GTPases that are required for vesicular transport. The cellular distribution of CRLR and RAMP1 will be defined in the intestine to ascertain whether the proteins are suitably located to comprise a functional CGRP receptor.
Aim 2 will define importance of beta-arrestins in mediating endocytosis and desensitization of NK1R and CRLR/RAMP1 in cell lines by expressing dominant negative mutants. The role of Beta-arrestins in terminating the inflammatory effects of SP and CGRP will be examined in vivo using Beta-arrestins 1 and 2 knockout animals.
Aim 3 will investigate novel mechanisms that account for the potentiating proinflammatory interactions between CGRP and SR The capacity of CGRP to inhibit the degradation of SP by NEP and thereby potentiate its proinflammatory effects will be evaluated in vitro and in the intact animal. Preliminary observations have identified a novel mechanism by which the SP induces sequestration of beta-arrestins into endosomes, thereby depleting cytosolic pools of beta-arrestins and attenuating endocytosis and desensitization of other receptors. The importance of this sequestration in potentiating CGRP signaling will be examined to determine whether this process could also account for the potentiating interactions of SP and CGRP. Together, the results of these experiments will provide new insights into peptide signaling, generate novel information about mechanisms of intestinal inflammation, and suggest new strategies for therapy.
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