GRK6 is a member of the GRK family of proteins that were initially identified as kinases responsible for the desensitization and downregulation of GPCRs. In this report, we identified GRK6 as an important molecule that promotes efficient apoptotic cell engulfment by several types of macrophages. A deficiency of this molecule resulted in an autoimmune-like disease and splenic iron accumulation in these mice. GRK6-mediated engulfment was largely independent of two well-established intracellular engulfment pathways, DOCK180/ELMO/Rac1 and GULP/Rac1, but did require GIT1 and the phosphorylation of radixin and moesin.
Both GRK2 and GRK6 expressions enhanced apoptotic engulfment. However, the promotion of apoptotic cell engulfment by GRK6 but not GRK2 depends on its kinase activity. A previous report showed that GRK2 positively regulated integrin-dependent epithelial cell migration in a kinase activity-independent manner, which was orchestrated by GIT120
. The function of GRK2 in cell migration was regulated by its phosphorylation at certain tyrosine and serine residues20
, which led to the interaction of GRK2 with GIT1. This interaction modulates the Rac1/PAK/MEK/ERK1/2 pathway in response to sphingosine-1-phosphate and adhesion20
. In contrast, the GRK6–GIT1 complex did not significantly influence this pathway (Supplementary Fig. S8
). Considering these differences, the pro-migratory or pro-engulfmental signal transduction pathway mediated by GRK244
or GRK6 is regulated by distinct molecular mechanisms, although GIT1 is common to both the pathways. GRK2 was reported to directly phosphorylate ERM46
, whereas it did not undergo direct phosphorylation by GRK6. This may also contribute to the different signalling pathways by the two GRKs. The molecular mechanism by which GRK2 or GRK6 defines its specific signal transduction pathway will be an interesting theme for future studies.
With regard to the clearance of senescent red blood cells by splenic macrophages, the CD47-SHPS-1 regulatory system has been well studied47
. The CD47-SHPS-1 system defines self- and non-self recognition by macrophages, which generates the so-called ‘Don’t eat me signal’ that enables viable red blood cells to escape engulfment by phagocytes. In this study, we found that GRK6 promoted the engulfment of senescent red blood cells by red splenic macrophages and contributed to iron homoeostasis. A recent report40
showed that Spic-deficient mice selectively lost splenic red pulp macrophages. In these mice, iron deposition in their spleens was observed due to the inefficient engulfment of dead red blood cells. In spite of this iron deposition, Spic-deficient mice did not show any significant abnormalities in total iron homoeostasis, which was similar to GRK6-deficient mice. To date, studies on the diseases of iron metabolism have usually focused on the proteins that directly alter iron transport, such as iron transporters/exporters, receptors for iron-binding proteins or the regulators of the activities of these transporters and receptors. Our study provides a new model of human iron disorders in the spleen. GRK6 may be a candidate gene that is responsible for iron overload in some patients.
In summary, we revealed that GRK6 was involved in apoptotic cell clearance by two independent subsets of macrophages in the spleen. GRK6 greatly contributed to removing unnecessary B cells by splenic white pulp macrophages and removing senescent red blood cells by splenic red pulp macrophages. However, we cannot rule out the possibility that some unidentified roles for GRK6, other than engulfment, influence the phenotypes of GRK6-deficient mice, as our experiments were performed using GRK6 systemic knockout mice. Thus, to conclude that the phenotypes observed here were due to engulfment defects by GRK6-deficient macrophages, it will be necessary to examine the phenotypes of GRK6-deficient mice after transplanting bone marrow cells from WT mice and vice versa.