This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. G-protein coupled receptor (GPCR) activation by neuropeptides is terminated by either cell-surface peptidase activity or receptor desensitization, and thereby prevents uncontrolled signaling that may cause dysfunction and disease. Cell-surface peptidases such as neutral endopeptidase (NEP) degrade extra-cellular neuropeptides and terminate their biological actions. For example, NEP degrades substance P (SP) and terminates inflammation by limiting the activation of the neurokinin-1 receptor (NK1R). Alternatively, activated GPCR signaling is terminated by desensitization, a process that entails uncoupling of heterotrimeric G-proteins and endocytosis of the receptor-agonist complex into endosomes where the acidic environment of the endosomes facilitates dissociation of the receptor and agonist. While the receptor is sorted to either recycling or degradative pathways by poorly understood mechanisms, the ultimate fate of the agonist after endocytosis is unknown. Endothelin-converting enzyeme-1 (ECE-1) is a member of the M-13 family of endopeptidases that also includes NEP. While NEP is confined to the plasma membrane, ECE-1 has a broad subcellular distribution that includes both the plasma membrane and endosomes. The primary natural substrate of ECE-1 expressed at the plasma membrane is big endothelin (big-ET). Big-ET is hydrolyzed by ECE-1 at the plasma membrane to form endothelin-1 (ET), a potent vasoconstrictor. While ECE-1 hydrolyzes a variety of neuropeptides such as substance-P and bradykinin in in-vitro experiments, the biological significance of this activity is unknown. Furthermore, the role of ECE-1 contained in endosomes has not been explored.
The aim of this project is to study the role of ECE-1 in GPCR signaling. Activation of GPCRs results in endocytosis of the receptor and agonist into acidified endosomes. We propose that endopeptidases such as ECE-1 are also present in these acidified endosomes where they degrade the neuropeptide agonists and thereby control receptor recycling. Mass spectrometry will allow for characterization of endosomal ECE-1 activity and determine its substrate specificity.
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