GRK6 deficiency in mice causes autoimmune disease due to impaired apoptotic cell clearance.

Efficient engulfment of apoptotic cells is critical for maintaining tissue homoeostasis. When phagocytes recognize 'eat me' signals presented on the surface of apoptotic cells, this subsequently induces cytoskeletal rearrangement of phagocytes for the engulfment through Rac1 activation. However, the intracellular signalling cascades that result in Rac1 activation remain largely unknown. Here we show that G-protein-coupled receptor kinase 6 (GRK6) is involved in apoptotic cell clearance. GRK6 cooperates with GIT1 to activate Rac1, which promotes apoptotic engulfment independently from the two known DOCK180/ELMO/Rac1 and GULP1/Rac1 engulfment pathways. As a consequence, GRK6-deficient mice develop an autoimmune disease. GRK6-deficient mice also have increased iron stores in splenic red pulp in which F4/80(+) macrophages are responsible for senescent red blood cell clearance. Our results reveal previously unrecognized roles for GRK6 in regulating apoptotic engulfment and its fundamental importance in immune and iron homoeostasis.

Determining the activation of rho as an index of receptor coupling to G12/13 proteins.

Heterotrimeric G proteins are composed of α, ß, and γ subunits. G proteins can be activated by a large number of cell-surface hepathelical receptors and can transduce signals from these receptors to various intracellular signaling molecules. When G protein-coupled receptors are bound by their cognate ligand, interaction with specific subtypes of G protein leads to dissociation of the α subunit of the heterotrimeric G protein from the ßγ dimer, and both Gα-GTP and Gßγ are capable of initiating their own signal transduction pathways. G proteins are functionally divided into four groups based on the nature of α subunit into G(s), G(i), G(q), and G12 families. The members of the G12 subfamily are G12 and G13. Increasing evidence indicates that G12/13 proteins play critical roles in various physiological functions. G12 and G13 regulate the small GTPase Rho through modulation of guanine nucleotide exchange factor (RhoGEF) activity to regulate various cellular responses, such as cytoskeletal changes and cell growth. Therefore, Rho activity can often represent a sensitive marker of G12/13 activity. Here, we describe the Rho activation assay to monitor the activity of G12/13 proteins.