title: NMR study of ligand release from asialoglycoprotein receptor under solution conditions in early endosomes.
abstract: 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 Ca(2+)  binding sites (sites 1, 2 and 3), with Ca(2+)  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 receptor-ligand 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 Ca(2+)  and ligands to ASGP-R CRD by NMR. We demonstrate that sites 1 and 2 of ASGP-R are high affinity Ca(2+)  binding sites, site 3 is low affinity, and that Ca(2+)  ions bind to sites 1 and 2 cooperatively. The pH and Ca(2+)  concentration dependences of Ca(2+)  binding states indicated that early endosome conditions favor apo-ASGP-R CRD, allowing ligand release. Our results elucidated that the cooperative binding mode of Ca(2+)  makes it possible for ASGP-R to be more sensitive to Ca(2+)  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 Ca(2+)  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.
authors.affiliation: Research and Development Department, Japan Biological Informatics Consortium, Tokyo, Japan.
authors.forename: Takuo
authors.initials: T
authors.lastname: Onizuka
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Shimizu
authors.forename: Yuka
authors.initials: Y
authors.lastname: Moriwaki
authors.forename: Takayuki
authors.initials: T
authors.lastname: Nakano
authors.forename: Shozo
authors.initials: S
authors.lastname: Kanai
authors.forename: Ichio
authors.initials: I
authors.lastname: Shimada
authors.forename: Hideo
authors.initials: H
authors.lastname: Takahashi
mesh_terms: D000595:Amino Acid Sequence; D037263:Asialoglycoprotein Receptor / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism; D002942:Circular Dichroism; D011992:Endosomes / Q000378:metabolism; D005690:Galactose / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D008024:Ligands; D008958:Models, Molecular; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D017434:Protein Structure, Tertiary; D021122:Protein Subunits; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D012996:Solutions; D020349:Surface Plasmon Resonance
chemical_list: D037263:Asialoglycoprotein Receptor; D008024:Ligands; D021122:Protein Subunits; D011994:Recombinant Proteins; D012996:Solutions; D002118:Calcium; D005690:Galactose