Date of Award
Doctor of Philosophy (PhD)
CXCR4 is a G protein-coupled receptor (GPCR) that binds to the chemokine, stromal cell-derived factor-1 (SDF-1alpha; a.k.a. CXCL12). The SDF-1alpha/CXCR4 signaling axis plays an essential role during embryogenesis in the development of the heart, brain and vasculature and in the adult mediating immune cell trafficking and stem cell homing to the bone marrow. Dysregulation of SDF-1alpha/CXCR4 signaling is linked to several pathological conditions, including cardiovascular disease, immunological disorders as well as cancer growth and metastasis. However, the mechanisms that govern CXCR4 signaling remain poorly understood. In this dissertation project, we attempt to further our understanding of the molecular mechanisms that regulate CXCR4 signaling.
CXCR4 signaling is tightly controlled by a complex series of events that rapidly terminates its signaling. Activated CXCR4 is rapidly phosphorylated and ubiquitinated by the E3 ubiquitin ligase AIP4 at the plasma membrane. Ubiquitinated CXCR4 is rapidly internalized onto early endosomes and targeted for lysosomal degradation, giving rise to long-term attenuation of signaling. The ubiquitin moiety on CXCR4 serves as a sorting signal for entry into the endosomal sorting complex required for transport (ESCRT) pathway. This pathway consists of four different protein complexes (ESCRT-0, I, II and III), plus several accessory factors, that act in a sequential and coordinated manner to target proteins for lysosomal degradation. Although CXCR4 is targeted into the ESCRT pathway, mechanistic insight by which this occurs remains poorly defined. In previous work from our laboratory, it was shown that adaptor protein arrestin-2 interacts with AIP4 to regulate endosomal sorting of CXCR4 into the degradative pathway. However, the precise mechanism by which arrestin-2 performs this function remains unknown.
We set out to determine how arrestin-2 integrates with the sorting machinery on endosomes to control the amount of CXCR4 that is degraded. We show that arrestin-2 interacts with ESCRT-0 protein STAM-1. ESCRT-0 consists of two proteins: signal-transducing adaptor molecule (STAM) and hepatocyte growth factor-regulated tyrosine kinase substrate (HRS). It is the first complex that recognizes ubiquitinated CXCR4 and targets it for lysosomal degradation. We show that depletion of STAM-1 by siRNA and selective disruption of the STAM-1/arrestin-2 interaction accelerates agonist promoted degradation of CXCR4, suggesting that STAM-1 via its interaction with arrestin-2 negatively regulates CXCR4 endosomal sorting. Interestingly, disruption of this interaction also blocks agonist promoted ubiquitination of HRS, the other ESCRT-0 protein, but not ubiquitination of CXCR4 and STAM-1, suggesting that arrestin-2 via its interaction with STAM-1 mediates ubiquitination of HRS. We propose a model, whereby arrestin-2 initially recruits CXCR4 to the ESCRT machinery and subsequently interacts with ESCRT-0 to regulate its sorting function, thereby ultimately controlling the amount of CXCR4 that is degraded.
Here, we also report novel roles for AIP4 and STAM-1 in CXCR4 signaling, which are different from their roles in CXCR4 trafficking. Treatment of cells with siRNA against AIP4 and STAM-1 attenuates CXCR4-induced activation of extracellular regulated kinase 1 and 2 (ERK-1/2). We show that STAM-1 via its SH3 domain interacts with the proline-rich region (PRR) in AIP4. AIP4 mediates STAM-1 ubiquitination and expression of AIP4-C830A, a catalytically inactive mutant that fails to ubiquitinate STAM-1, and an AIP4 mutant (AIP4-delta PRR) that shows poor binding to STAM-1 fail to enhance CXCR4-induced ERK-1/2 activation, suggesting that interaction with STAM-1 as well as ubiquitination activity of AIP4 are important for CXCR4-induced ERK-1/2 activation. Remarkably, a discrete subpopulation of AIP4 and STAM-1 co-localize with CXCR4 at the plasma membrane and with caveolin-1, a protein that is enriched in caveolae (a specialized lipid raft). Disrupting caveolae using caveolin-1 siRNA or nystatin, a cholesterol-depleting agent, significantly attenuated CXCR4-induced ERK-1/2 activation. Based upon our data, we propose that AIP4-mediated ubiquitination of STAM-1 in caveolae coordinates CXCR4 activation of ERK-1/2 signaling.
Taken together, our study has provided novel insight into the regulation of CXCR4 both at the levels of signaling as well as trafficking. We provide novel mechanistic insight into the role of arrestin-2 in targeting CXCR4 into the degradative pathway and we have also identified a novel function for AIP4 and STAM-1 in CXCR4 signaling.
Malik, Rohit, "Molecular Mechanisms Regulating Chemokine Receptor CXCR4 Signaling and Trafficking" (2012). Dissertations. 309.
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Copyright © 2012 Rohit Malik