The role of ubiquitin in GPCR signaling

ABSTRACT The G protein-coupled receptor (GPCR) C-X-C chemokine receptor 4 (CXCR4) and its cognate ligand CXCL12 have been linked to endothelial cell migration, a necessary part of angiogenesis in health and disease. Specifically, arterial expression of CXCR4 promotes neovascularization of the heart following myocardial

infarction, reducing damage and leading to recovery, yet the mechanisms that control CXCR4 signaling remain poorly understood. Better understanding of CXCR4 signaling could aid in developing improved therapeutics for cardiovascular disease. CXCR4 abundance and signaling is regulated by the post-translational modification

termed ubiquitination, but the mechanisms remain poorly understood. The objective of this project is to better understand how ubiquitination governs CXCR4 signaling and trafficking. Stimulation with CXCL12 induces ubiquitination of C-terminal lysine residues on CXCR4, serving as a sorting signal into the multi-vesicular

body/endosomal sorting complexes required for transport (MVB/ESCRT) pathway for subsequent degradation in lysosomes leading to downregulation of signaling. In addition, agonist activation of CXCR4 promotes ubiquitination of STAM1, a component of the ESCRT-0 complex. Ubiquitination of STAM1 requires the

endocytic adaptor proteins b-arrestin1 and is implicated in regulating CXCR4 trafficking and signaling. Despite this pivotal role very little is known concerning STAM1 ubiquitination. Preliminary data suggest that b-arrestin1 increases ubiquitination of STAM1 by the E3 ubiquitin ligase AIP4, consistent with b-arrestin1 serving as an

adaptor for STAM1 ubiquitination. Mass spectroscopy analysis revealed that STAM1 is modified by ubiquitin at several lysine residues and with potentially multiple types of ubiquitin linkages. Remarkably, one of the ubiquitin linkages corresponded to linear or Met1-linked ubiquitin chains, a linkage type only previously known

to form by the E3 ligase complex LUBAC in the context of NFkB signaling. Further biochemical studies suggest that b-arrestin1 is required for M1-linked linear ubiquitin chain formation at a specific lysine residue on STAM1 by AIP4. Based on the preliminary data, we hypothesize that b-arrestin1 coordinates lysine selection by AIP4

to mediate linear ubiquitination of STAM1. To test this hypothesis, we will use several biochemical, molecular and biophysical approaches (Aim 1) to identify and characterize site-specific linear ubiquitination of STAM1 by b-arrestin1 via AIP4 and (Aim 2) to determine the role of linear poly-ubiquitin chains on GPCR trafficking and

signaling. At the conclusion of the project, we will have identified an unexpected role for M1-linked ubiquitin chains in GPCR signaling and trafficking. This project will be carried out at the Medical College of Wisconsin under the mentorship of Dr. Adriano Marchese, an expert on the role of ubiquitin on GPCR signaling and

trafficking. The sponsor lab and the overall scientific community at the Medical College of Wisconsin provides an outstanding environment to successfully complete the proposed studies.