Analysis of CCR2 splice variant expression patterns and functional properties

Abstract

Background C–C motif chemokine receptor 2 (CCR2), the main receptor for monocyte chemoattractant protein-1 (MCP-1), is expressed on immune cells, including monocytes, macrophages, and activated T cells, and mediates cell migration toward MCP-1 in inflammation-related diseases. The CCR2 gene encodes two isoforms: CCR2A and CCR2B. The CCR2B open reading frame is localized in a single exon, similar to other chemokine receptors, and CCR2A and CCR2B feature different amino acid sequences in their C-terminal intracellular loops due to alternative splicing. Most biochemical studies on CCR2-related cellular responses in the immune system have focused on CCR2B, with few reports focused on CCR2A. Understanding the functional properties of CCR2A in cellular responses may elucidate the roles played by MCP-1 and CCR2 in pathophysiological responses. Results CCR2 gene expression analysis in several cell types revealed that most adherent cells only expressed CCR2A, whereas CCR2B expression was dominant in monocytic cells. The C-terminal Helix 8 region of CCR2A contains few basic amino acids, which may be unfavorable for cell surface localization, as confirmed with the HiBiT assay. CCR2B contains many C-terminal Ser/Thr residues, similar to other chemokine receptors, which may be phosphorylated by G protein–coupled receptor kinases (GRKs) to promote β-arrestin recruitment and subsequent endocytosis. By contrast, CCR2A contains few C-terminal Ser/Thr residues, which are unlikely to be phosphorylated by GRKs. CCR2A localized on the cell surface is resistant to internalization, despite the interaction between Gβ and GRKs induced by ligand binding with CCR2A. CCR2A induced cellular responses at a relatively higher degree than CCR2B, although both receptors mediated signaling events through Gαq and Gαi. HeLa cells lacking CCR2A showed slowed growth compared with parent cells, regardless of MCP-1 stimulation, and their chemotactic activity toward MCP-1, in addition to basal motility, was significantly impaired. Conclusion MCP-1 and CCR2 may play pivotal roles in cancer progression by recruiting macrophages into cancer tissue. This study demonstrates that CCR2A but not CCR2B is expressed in solid cancer–derived cells. CCR2A is resistant to internalization by β-arrestin due to a distinct C-terminal region from CCR2B, which enhances MCP-1-stimulated responses, indicating that CCR2A may play essential roles in solid cancer progression.

The N-terminus of CXCR4 splice variants determines expression and functional properties

“…Expression of all constructs was driven by the ubiquitin C promoter. Finally, the best signals were observed in combination with CXCR4-SmBiT and LgBiT-mini G proteins, as shown in other chemokine signaling studies [ 31 ]. HEK293 cells expressing CXCR4 variant 2-SmBiT and each LgBiT-mini G protein were stimulated with CXCL12, and luciferase activity was measured in real-time.…”

“…However, chemokine-stimulated Ca 2+ influx was not detected even though the NanoBiT-based Ca 2+ assay is very sensitive. Regardless of mRNA expression level, the endogenous CXCR4 protein level seems to be sufficient to induce ERK phosphorylation but not to trigger intracellular Ca 2+ increase, which is consistent with cellular response to monocyte chemoattractant protein-1 (MCP-1) activation of endogenous CCR2 in HEK293 cells [ 31 ]. In other words, ERK phosphorylation is the most sensitive response to GPCR activation.…”

Efficient Redirection of NK Cells by Genetic Modification with Chemokine Receptors CCR4 and CCR2B

“…The chemokine receptors CCR4 and CCR2B were cloned from human peripheral mononuclear cells into a lentiviral expression vector. Thereby, the CCR2 isoform CCR2B was isolated and used throughout all experiments, as CCR2B is the predominant isoform expressed on monocytes [ 52 , 53 ].…”

Human inherited CCR2 deficiency underlies progressive polycystic lung disease

“…CCL-2-stimulated Ca 2+ mobilization was rescued in cells expressing WT CCR2 but not in cells expressing variants from the patients or a functional negative control variant lacking a G protein-binding site (p.K227_R243del) ( Figure 3B ). By contrast, all but one homozygous variant in our in-house cohort and gnomAD rescued CCL-2-stimulated Ca 2+ mobilization; the exception was p.L133F, which was isomorphic for CCR2B, the main isoform expressed in leukocytes, 39 , 40 and hypomorphic for CCR2A ( Figure 3B ). CCL-13, another CCR2 ligand, 41 did not stimulate Ca 2+ mobilization in CCR2 KO THP-1 cells after vector-mediated expression of any patient variant ( Figure 3C ).…”