Chemokine-Like Receptor 1 (CMKLR1), a G Protein Coupled Receptor Expressed On Proinflammatory Monocytes in Arthritis, Is Negatively Regulated by GRK3

D. Stephen Serafin¹, Roman Timoshchenko¹, Marcus W. McGinnis² and Teresa K. Tarrant³, ¹Thurston Arthritis Research Center, University of North Carolina at Chapel Hill, Chapel Hill, NC, ²Thurston Arthritis Research Center, University of North Carolina Sch of Med, Chapel Hill, NC, ³Medicine/Rheumatology, UNC School of Medicine, Chapel Hill, NC

Meeting: 2012 ACR/ARHP Annual Meeting

Keywords: chemokines, inflammation and monocytes

Session Information

Title: Cytokines, Mediators, and Gene Regulation

Session Type: Abstract Submissions (ACR)

Background/Purpose: Chemokine-like receptor 1 (CMKLR1) is a G protein coupled receptor (GPCR) expressed by inflammatory monocytes that are pathogenic in human diseases such as psoriasis and lupus. Its cognate ligand, chemerin, is a chemoattractant for invading inflammatory cells and is present in the synovial lining of rheumatoid arthritis (RA), osteoarthritis (OA), and psoriatic arthritis (PsA) patients. In OA, chemerin is thought to contribute to both the extracellular matrix breakdown of cartilage as well as joint inflammation.

CMKLR1 is a newly discovered GPCR, which as a class of receptors are phosphorylated by G-protein receptor kinases (GRKs) to turn off GPCR signaling. Due to the significant role of chemerin/CMKLR1 interaction in human inflammatory arthritis, we chose to examine the unknown regulatory mechanism of CMKLR1 by GRKs.

Methods: Isoforms GRK3 and GRK2 were separately knocked down in human monocytic THP-1 cells using shRNA and confirmed via quantitative real time PCR. Migration to chemerin (100 ng/mL) was measured by fluorescence emission through the BD Falcon 96-Multiwell Insert System or flow cytometry through 5 µM Transwells over 3 hrs.

A modified Tango assay was used to measure β-arrestin recruitment to CMKLR1. HTLA cells were over expressed with CMKLR1 and GRK2 or GRK3 via calcium phosphate precipitation. Cells were stimulated with 1 µM of chemerin and after 24 hours, luminescence was measured on a Trilux plate reader.

Results: CMKLR1 recruits β-arrestin in a ligand-dependent dose response. β-arrestin recruitment to CMKLR1 is mediated preferentially by GRK3 as compared to GRK2. THP-1 cells deficient in GRK3 showed enhanced chemotaxis to chemerin when compared to control cells.

Conclusion: We conclude that the chemerin/ CMKLR1 axis plays an important role in targeting monocytes to sites of inflammation and is negatively regulated by GRK3.

Disclosure:

D. S. Serafin,
None;

R. Timoshchenko,
None;

M. W. McGinnis,
None;

T. K. Tarrant,
None.

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