Functional domains of the very low density lipoprotein receptor: molecularanalysis of ligand binding and acid-dependent ligand dissociation mechanisms

The very low density lipoprotein (VLDL) receptor is closely related in stru cture to the low density lipoprotein receptor. The ectodomain of these endo cytic receptors is composed of modules which include clusters of cysteine-r ich class A repeats, epidermal growth factor (EGF)-like repeats, tyrosine-t ryptophan-threonine-aspartic acid (YWTD) repeats and an O-linked sugar doma in. To identify important functional regions within the ectodomain of the V LDL receptor, we produced a mutant receptor in which the EGF, YWTD and O-li nked sugar domains were deleted. Cells transfected with the mutant receptor were able to bind and internalize I-125-labeled receptor associated protei n (RAP), In contrast to the wild-type receptor, however, RAP did not dissoc iate from the mutant receptor and consequently was not degraded. Immunofluo resence 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 wildtype receptor, RAP fluorescence co-localized with lysosomal markers. Thus this deleted region is responsible for ligand unco upling within the endosomes, To identify regions responsible for ligand rec ognition, soluble receptor fragments containing the eight cysteine-rich cla ss A repeats were produced. I-125-RAP and I-125-labeled urokinase-type plas minogen activator:plasminogen activator inhibitor type I(uPA:PAI-1) complex es bound to the soluble fragment with K-D,K-app values of 0.3 and 14 nM, re spectively, Deletion analysis demonstrate that high affinity RAP binding re quires the first four cysteine-rich class A repeats (L1-4) in the VLDL rece ptor while the second repeat (L2) appears responsible for binding uPA:PAI-1 complexes. Together, these results confirm that ligand uncoupling occurs v ia 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 p ortion of the molecule.