The role of the cytoplasmic domain of the macrophage scavenger receptor MARCO in adhesion and uptake

The macrophage receptor with collagenous structure (MARCO) is a class A scavenger receptor. Class A scavenger receptors are multifunctional type II transmembrane glycoproteins that have roles in modified lipoprotein uptake, innate immunity, and macrophage adhesion. It has been shown that the extracellular cysteine-rich domain of MARCO is important for ligand binding, but the role of the cytoplasmic N-terminal domain has not yet been characterised. The aim of this study was to investigate the role of the cytoplasmic domain in cell adhesion and motility, ligand binding and uptake. Two truncated forms of human MARCO were used in this study: N- MARCO lacks the entire cytoplasmic domain; Nmyr MARCO includes the membrane-proximal putative myristoylation site, but lacks the rest of the cytoplasmic domain. The constructs were expressed in transiently transfected HEK 293T cells. The Nmyr MARCO mutant had reduced cell surface expression, an effect that was even more apparent in cells transfected with NMARCO. This indicates that the putative myristoylation site is not obligatory for cell surface expression of MARCO, but increases cell surface expression of the protein. When bound to ligand, the two mutants demonstrated a significant increase in cell surface expression, while the cell surface expression of the full-length MARCO remained unchanged. This indicates that the cytoplasmic tail affects membrane trafficking and the regulation of cell surface expression. Maleylated proteins are ligands for scavenger receptor A and other scavenger receptors. This study demonstrates that maleylated bovine serum albumin (MalBSA) is also a ligand for MARCO and this modified protein was used to study MARCO-mediated adhesion and ligand binding. The results show that the cytoplasmic domain of MARCO is not required for binding to MalBSA coated particles. Interestingly, the Nmyr MARCO mutant showed a decreased ability to bind soluble MalBSA compared to the full-length protein indicating that the cytoplasmic domain may be necessary for endocytosis. The cytoplasmic domain of MARCO appeared to be absolutely required for adhesion to tissue culture plastic, anti-MARCO antibody PLK-1 coated surfaces and suspension grade plastic, and a lesser extent to MalBSA coated surfaces. MARCO mediated adhesion was shown not to require the presence of serum and divalent cations. Studies performed with murine primary resident peritoneal macrophages on a MalBSA coated surface indicated that MARCO is responsible for increased chemokinesis. The results of this study show that the cytoplasmic domain of MARCO affects cell surface expression, endocytosis and adhesion. Further research is needed to determine if and how the cytoplasmic domain on MARCO participates in signalling required for these processes and if it affects cell motility.