RESUMEN
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.
Asunto(s)
Ácidos/farmacología , Receptores de LDL/química , Receptores de LDL/metabolismo , Regulación Alostérica , Animales , Células CHO , Células COS , Cricetinae , Cartilla de ADN , ADN Complementario , Endocitosis/fisiología , Factor de Crecimiento Epidérmico/química , Factor de Crecimiento Epidérmico/genética , Receptores ErbB/análisis , Receptores ErbB/química , Receptores ErbB/genética , Técnica del Anticuerpo Fluorescente , Eliminación de Gen , Humanos , Radioisótopos de Yodo , Ligandos , Mutagénesis/fisiología , Fragmentos de Péptidos/metabolismo , Inhibidor 1 de Activador Plasminogénico/metabolismo , Inhibidor 1 de Activador Plasminogénico/farmacología , Unión Proteica/efectos de los fármacos , Estructura Terciaria de Proteína , Ensayo de Unión Radioligante , Receptores de LDL/genética , Proteínas Recombinantes de Fusión/metabolismo , Transfección , Transferrina/análisis , Activador de Plasminógeno de Tipo Uroquinasa/metabolismo , Activador de Plasminógeno de Tipo Uroquinasa/farmacologíaRESUMEN
Over the past several years, national coalitions and multi-hospital systems have been expanding and increasing in popularity. These organizations have offered the member hospitals services and resources to enhance the competitive position of the member hospitals. As these coalitions have grown, they have approached children's hospitals but, in most cases, to be an affiliate of one of the general acute care hospitals. The services of these coalitions are designed to enhance the general acute care centers and have not been refined to address the needs of children's specialty centers. As children's hospitals around the country assessed how they should work with these national coalitions, they were faced with several challenges. These coalitions did pose a potential competitive threat to the children's hospitals. But, at the same time, the coalitions did not offer services to the children's hospitals that would justify membership and the large outlay in dues. Faced with this dilemma, a group of children's hospitals came together to develop a formal national coalition for children's hospitals. This case provides an excellent example of and the opportunity to explore the implementation of a collaborative strategy designed to create competitive advantage for each of the collaborators.