NMR study of ligand release from asialoglycoprotein receptor under solution conditions in early endosomes.

Asialoglycoprotein receptor (ASGP-R) is an endocytic C-type lectin receptor in hepatocytes that clears plasma glycoconjugates containing a terminal galactose or N-acetylgalactosamine. The carbohydrate recognition domain (CRD) of ASGP-R has three Ca2+ binding sites (sites 1, 2 and 3), with Ca2+ at site 2 being directly involved in ligand binding. Following endocytosis, the ligands are released from ASGP-R in endosomes to allow receptor recycling to the cell membrane. Although dissociation of the receptorligand complex is mediated by the acidic environment within the mature endosomes, many of these complexes also dissociate in the early time of endocytosis, where pH is approximately neutral. To investigate the mechanism of ligand release from ASGP-R in early endosomes, we examined the binding mode of Ca2+ and ligands to ASGP-R CRD by NMR. We demonstrate that sites 1 and 2 of ASGP-R are high affinity Ca2+ binding sites, site 3 is low affinity, and that Ca2+ ions bind to sites 1 and 2 cooperatively. The pH and Ca2+ concentration dependences of Ca2+ binding states indicated that early endosome conditions favor apo-ASGP-R CRD, allowing ligand release. Our results elucidated that the cooperative binding mode of Ca2+ makes it possible for ASGP-R to be more sensitive to Ca2+ concentrations in early endosomes, and plays an important role in the efficient release of ligand from ASGP-R. In our proposed mechanism, ASGP-R can rapidly release Ca2+ and its ligand even at nearly neutral pH. Sequence comparisons of endocytic C-type lectin receptors suggest that this mechanism is common in their family.