Systemic Glycosaminoglycan Clearance by HARE/Stabilin-2 Activates Intracellular Signaling

Scavenger receptors perform essential functions, critical to maintaining mammalian physiologic homeostasis by continuously clearing vast numbers of biomolecules from blood, interstitial fluid and lymph. Stabilin-2 (Stab2) and the Hyaluronic Acid Receptor for Endocytosis (HARE), a proteolytic isoform of Stab2, are important scavenger receptors responsible for the specific binding and internalization (leading to degradation) of 22 discrete molecules, macromolecular complexes and cell types. One-third of these ligands are glycosaminoglycans (GAGs). Full-length Stab2, but not HARE, mediates efficient phagocytosis of apoptotic cells and bacteria via binding to target surface ligands. HARE, the C-terminal half of Stab2, mediates endocytosis of all the known soluble ligands. HA was the first ligand identified, in 1981, prior to receptor purification or cloning. Seven other GAG ligands were subsequently identified: heparin, dermatan sulfate, chondroitin and chondroitin sulfates A, C, D and E. Synthetic dextran sulfate is also a GAG mimic and ligand. HARE signaling during HA endocytosis was first discovered in 2008, and we now know that activation of HARE/Stab2 signaling is stimulated by receptor-mediated endocytosis or phagocytosis of many, but not all, of its ligands. This review focuses on the HARE-mediated GAG activation of intracellular signaling, particularly the Extracellular Signal-Regulated Kinase 1/2 pathway.

Purification of A1 adenosine receptor–G-protein complexes: effects of receptor down-regulation and phosphorylation on coupling

We examined the effects of exposing A1 adenosine receptors (A1ARs) to an agonist on the stability and phosphorylation state of receptor–guanine nucleotide-binding regulatory protein (R–G-protein) complexes. Non-denatured recombinant human A1ARs extended on the N-terminus with hexahistidine (His6) and the FLAG (Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys) epitope (H/F) were purified to near homogeneity from stably transfected Chinese-hamster ovary (CHO)-K1 cells. Purified receptors have pharmacological properties similar to receptors in membranes. G-proteins were co-purified with 15±2% of H/F-A1AR unless receptor–G-protein (R–G) complexes were uncoupled by pre-treating cell membranes with GTP. By silver staining, purified A1AR–G-protein complexes contain receptors, G-protein α and β subunits and an unidentified 97 kDa protein. Pretreating intact cells with N6-cyclopentyladenosine (CPA) for 24 h decreased both the total number of receptors measured in membranes and the number of purified A1ARs by about 50%. In contrast, pretreating cells with CPA decreased the number of R–G complexes measured in membranes (54±6%) significantly less than it decreased the number of purified R–G complexes (78±3%) as detected by 125I-N6-(4-aminobenzyl)adenosine binding or by Western blotting Giα2. The effect of CPA to decrease the fraction of receptors purified as R–G complexes was not associated with any change in low-level A1AR phosphorylation (found on serine), or low-level phosphorylation of G-protein α or β subunits or the 97 kDa protein. These experiments reveal a novel aspect of agonist-induced down-regulation, namely a diminished stability of receptor–G-protein complexes that is manifested as uncoupling during receptor purification.

Mesangial cells possess an asialoglycoprotein receptor with affinity for human immunoglobulin A.

Asialoglycoprotein receptor (ASGP-R), a hepatic lectin involved in the clearance of galactose-terminal glycoproteins, is also present in extrahepatic tissues, but its expression in renal cells is not well established. This study examines the presence of ASGP-R in cultured mesangial cells (MC), key cells involved in the removal of macromolecules deposited in the glomerulus. The binding of asialo-orosomucoid (ASOR) to rat MC was saturable and galactose-specific. In addition, MC internalized and degraded ASOR in a Ca(2+)-dependent manner. Parallel studies were performed in a homologous system (human MC), obtaining similar binding curve and competition with unlabeled ASOR and carbohydrates. The purified receptor from rat MC consisted of two proteins (41 and 55 kD) with similar size to the hepatic receptor. Both subunits were detected by mRNA expression analysis (ratio 2:1). Because the hepatic receptor presents avidity for the carbohydrates of IgA1, a protein deposited in the glomerulus of patients with IgA nephropathy, the interaction of IgA1 with the mesangial ASGP-R was explored. As for the interaction with ASOR, catabolism of IgA1 by rat and human MC was Ca(2+)-dependent and was reduced with galactose. In addition, the interaction of ASOR with rat MC was partially inhibited by incubation with IgA1 and its desialylated form, but not by IgA2, as demonstrated in binding experiments and in receptor purification. It is concluded that MC possess ASGP-R specific for galactose residues of several glycoproteins, including IgA1. These data could be important for a better understanding of the pathogenesis of IgA nephropathy.