title: Functional domains of the very low density lipoprotein receptor: molecular analysis of ligand binding and acid-dependent ligand dissociation mechanisms.
abstract: The very low density lipoprotein (VLDL) receptor is closely related in structure to the low density lipoprotein receptor. The ectodomain of these endocytic receptors is composed of modules which include clusters of cysteine-rich class A repeats, epidermal growth factor (EGF)-like repeats, tyrosine-tryptophan-threonine-aspartic acid (YWTD) repeats and an O-linked sugar domain. To identify important functional regions within the ectodomain of the VLDL receptor, we produced a mutant receptor in which the EGF, YWTD and O-linked sugar domains were deleted. Cells transfected with the mutant receptor were able to bind and internalize (125)I-labeled receptor associated protein (RAP). In contrast to the wild-type receptor, however, RAP did not dissociate from the mutant receptor and consequently was not degraded. Immunofluoresence data indicated that once bound to the mutant receptor, fluorescent-labeled RAP co-localized with markers of the endosomal pathway, whereas, in cells expressing the wild-type receptor, RAP fluorescence co-localized with lysosomal markers. Thus this deleted region is responsible for ligand uncoupling within the endosomes. To identify regions responsible for ligand recognition, soluble receptor fragments containing the eight cysteine-rich class A repeats were produced. (125)I-RAP and (125)I-labeled urokinase-type plasminogen activator:plasminogen activator inhibitor type I (uPA:PAI-1) complexes bound to the soluble fragment with K(D, app) values of 0.3 and 14 nM, respectively. Deletion analysis demonstrate that high affinity RAP binding requires the first four cysteine-rich class A repeats (L1-4) in the VLDL receptor while the second repeat (L2) appears responsible for binding uPA:PAI-1 complexes. Together, these results confirm that ligand uncoupling occurs via an allosteric-type mechanism in which pH induced changes in the EGF and/or YWTD repeats alter the ligand binding properties at the amino-terminal portion of the molecule.
authors.affiliation: Department of Vascular Biology, Holland Laboratory, American Red Cross, Rockville Maryland 20855, USA.
authors.forename: I
authors.initials: I
authors.lastname: Mikhailenko
authors.forename: W
authors.initials: W
authors.lastname: Considine
authors.forename: K M
authors.initials: KM
authors.lastname: Argraves
authors.forename: D
authors.initials: D
authors.lastname: Loukinov
authors.forename: B T
authors.initials: BT
authors.lastname: Hyman
authors.forename: D K
authors.initials: DK
authors.lastname: Strickland
mesh_terms: D000143:Acids / Q000494:pharmacology*; D000494:Allosteric Regulation; D000818:Animals; D016466:CHO Cells; D019556:COS Cells; D006224:Cricetinae; D017931:DNA Primers; D018076:DNA, Complementary; D004705:Endocytosis / Q000502:physiology; D004815:Epidermal Growth Factor / Q000737:chemistry / Q000235:genetics; D066246:ErbB Receptors / Q000032:analysis / Q000737:chemistry / Q000235:genetics; D005455:Fluorescent Antibody Technique; D017353:Gene Deletion; D006801:Humans; D007457:Iodine Radioisotopes; D008024:Ligands; D016296:Mutagenesis / Q000502:physiology; D010446:Peptide Fragments / Q000378:metabolism; D017395:Plasminogen Activator Inhibitor 1 / Q000378:metabolism / Q000494:pharmacology; D011485:Protein Binding / Q000187:drug effects; D017434:Protein Structure, Tertiary; D011869:Radioligand Assay; D011973:Receptors, LDL / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014162:Transfection; D014168:Transferrin / Q000032:analysis; D014568:Urokinase-Type Plasminogen Activator / Q000378:metabolism / Q000494:pharmacology
chemical_list: D000143:Acids; D017931:DNA Primers; D018076:DNA, Complementary; D007457:Iodine Radioisotopes; D008024:Ligands; D010446:Peptide Fragments; D017395:Plasminogen Activator Inhibitor 1; D011973:Receptors, LDL; D011993:Recombinant Fusion Proteins; D014168:Transferrin; C036053:VLDL receptor; D004815:Epidermal Growth Factor; D066246:ErbB Receptors; D014568:Urokinase-Type Plasminogen Activator