C-X-C Motif Chemokine Receptor 3 Splice Variants Differentially Activate Beta-Arrestins to Regulate Downstream
Jeffrey S Smith1, Priya Alagesan1, Nimit K Desai1
1Department of Biochemistry (J.S.S., P.A., N.K.D., S.R.), Department of Pharmacology and Cancer Biology (T.F.P.), and Department of Medicine (J.-H.W., N.J.F., S.R.), Duke University Medical Center, Durham, NC 27710; Department of Pharmaceutical Sciences, Tohoku University, Japan (A.I.); and Japan Science and Technology Agency, Precursory Research for Embryonic Science and Technology (PRESTO), Japan (A.I.).
Abstract:
Biased agonism, the ability of different ligands for the same receptor to selectively activate some signaling pathways while blocking others, is now an established paradigm for G protein-coupled receptor signaling. One group of receptors in which endogenous bias is critical is the chemokine system, consisting of over 50 ligands and 20 receptors that bind one another with significant promiscuity. We have previously demonstrated that ligands for the same receptor can cause biased signaling responses. The goal of this study was to identify mechanisms that could underlie biased signaling between different receptor splice variants. The C-X-C motif chemokine receptor 3 (CXCR3) has two splice variants, CXCR3A and CXCR3B, which differ by 51 amino acids at its N-terminus. Consistent with an earlier study, we found that C-X-C motif chemokine ligands 4, 9, 10, and 11 all activated G i at CXCR3A, while at CXCR3B these ligands demonstrated no measurable G i or G s activity. β-arrestin (βarr) was recruited at a reduced level to CXCR3B relative to CXCR3A, which was also associated with differences in βarr2 conformation. βarr2 recruitment to CXCR3A was attenuated by both G protein receptor kinase (GRK) 2/3 and GRK5/6 knockdown, while only GRK2/3 knockdown blunted recruitment to CXCR3B. Extracellular regulated kinase 1/2 phosphorylation downstream from CXCR3A and CXCR3B was increased and decreased, respectively, by βarr1/2 knockout. The splice variants also differentially activated transcriptional reporters. These findings demonstrate that differential splicing of CXCR3 results in biased responses associated with distinct patterns of βarr conformation and recruitment. Differential splicing may serve as a common mechanism for generating biased signaling and provides insights into how chemokine receptor signaling can be modulated post-transcriptionally.
Insights
Differential splicing of C-X-C motif chemokine receptor 3 (CXCR3) generates biased signaling. CXCR3A and CXCR3B variants exhibit distinct G protein and beta-arrestin recruitment, impacting downstream signaling and transcriptional responses.
Area of Science:
- Pharmacology
- Molecular Biology
- Immunology
Background:
- Biased agonism is a key mechanism in G protein-coupled receptor (GPCR) signaling.
- Chemokine receptors, including C-X-C motif chemokine receptor 3 (CXCR3), exhibit promiscuous ligand binding and endogenous bias.
- CXCR3 exists as two splice variants, CXCR3A and CXCR3B, differing in their N-termini.
Purpose of the Study:
- To investigate the mechanisms underlying biased signaling between CXCR3 splice variants.
- To determine how differential splicing of CXCR3 influences G protein and beta-arrestin signaling pathways.
- To explore the functional consequences of CXCR3 splice variant diversity on downstream cellular responses.
Main Methods:
- Comparative analysis of G protein (Gi, Gs) activation by chemokine ligands at CXCR3A and CXCR3B.
- Assessment of beta-arrestin (βarr) recruitment and conformation to CXCR3 variants using knockdown of G protein receptor kinases (GRKs).
- Evaluation of extracellular regulated kinase 1/2 (ERK1/2) phosphorylation and transcriptional reporter activation in response to splice variant activation and βarr knockout.
Main Results:
- Chemokine ligands activated Gi at CXCR3A but showed no significant Gi or Gs activity at CXCR3B.
- Reduced beta-arrestin recruitment to CXCR3B compared to CXCR3A, with distinct βarr2 conformations.
- Differential GRK-mediated regulation of βarr recruitment and opposing effects on ERK1/2 phosphorylation by βarr knockout for each variant.
- CXCR3 splice variants differentially activated transcriptional reporters.
Conclusions:
- Differential splicing of CXCR3 generates functionally distinct receptor variants (CXCR3A and CXCR3B) that exhibit biased signaling.
- Distinct patterns of βarr conformation and recruitment underlie the biased signaling observed between CXCR3 splice variants.
- Post-transcriptional modulation via differential splicing may be a common strategy for generating biased signaling in the chemokine system.
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