Low-density-lipoprotein-receptor-related protein (LRP) interacts with a GTP-binding protein

The low-density-lipoprotein-receptor-related protein (LRP) binds and internalizes numerous ligands, including lipoproteins, proteinase–inhibitor complexes and others. We have shown previously that LRP-mediated ligand internalization is dependent on cAMP-dependent protein kinase (PKA) activity. Here, we investigated whether ligation of LRP increases the intracellular cAMP level and PKA activity via a stimulatory GTP-binding protein. Treatment of LRP-expressing cell lines with the LRP ligands lactoferrin or urokinase-type plasminogen activator caused a significant elevation in cAMP and stimulated PKA activity in a dose-dependent manner. Addition of the 39 kDa receptor-associated protein (RAP), an antagonist for ligand interactions with LRP, blocked the lactoferrin-induced increase in PKA activity, demonstrating a requirement for ligand binding to LRP. Incubation of cell membrane fractions with lactoferrin increased GTPase activity in a time- and dose-dependent manner, and treatment with LRP ligands suppressed cholera-toxin-mediated ADP-ribosylation of the Gsα subunit of a heterotrimeric G-protein. Affinity precipitation of LRP with RAP resulted in co-precipitation of two isoforms of Gsα from detergent extracts. We thus conclude that LRP is a signalling receptor that associates directly with a stimulatory heterotrimeric G-protein and activates a downstream PKA-dependent pathway.

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Three Complement-Type Repeats of the Low-Density Lipoprotein Receptor-Related Protein Define a Common Binding Site for RAP, PAI-1, and Lactoferrin

Blood ◽

10.1182/blood.v92.9.3277 ◽

1998 ◽

Vol 92 (9) ◽

pp. 3277-3285 ◽

Cited By ~ 27

Author(s):

Brian Vash ◽

Neil Phung ◽

Sima Zein ◽

Dianne DeCamp

Keyword(s):

Low Density Lipoprotein ◽

Density Lipoprotein ◽

Low Density ◽

Lipoprotein Receptor ◽

Dependent Manner ◽

Related Protein ◽

Low Density Lipoprotein Receptor ◽

Density Lipoprotein Receptor ◽

Lipoprotein Receptor Related Protein ◽

Pai 1

Abstract The low-density lipoprotein receptor-related protein (LRP) is a 600-kD scavenger receptor that binds a number of protein ligands with high affinity. Although some ligands do not compete with each other, binding of all is uniformly blocked by the 39-kD receptor-associated protein (RAP). RAP is normally found in the endoplasmic reticulum and seems to function as a chaperone for LRP. To identify the binding sites for RAP, lactoferrin, and plasminogen activator inhibitor-1 (PAI-1), a bacterial expression system has been developed to produce soluble LRP fragments spanning residues 783-1399. These residues overlap most of the CNBr fragment containing the second cluster of complement-type repeats (C). Solid phase binding assays show that 125I-RAP binds to fragments containing three successive complement-type repeats: C5-C7. PAI-1 and lactoferrin bind to the same fragments. A fragment containing C5-C7 also blocks uptake and degradation of 125I-RAP by fibroblasts in a concentration-dependent manner. Binding competition experiments show that RAP, PAI-1, and lactoferrin each inhibit the binding of the others, suggesting that at this site in LRP, RAP acts as a competitive, rather than an allosteric, inhibitor of PAI-1 and lactoferrin binding. © 1998 by The American Society of Hematology.

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Three Complement-Type Repeats of the Low-Density Lipoprotein Receptor-Related Protein Define a Common Binding Site for RAP, PAI-1, and Lactoferrin

Blood ◽

10.1182/blood.v92.9.3277.421k50_3277_3285 ◽

1998 ◽

Vol 92 (9) ◽

pp. 3277-3285 ◽

Cited By ~ 1

Author(s):

Brian Vash ◽

Neil Phung ◽

Sima Zein ◽

Dianne DeCamp

Keyword(s):

Low Density Lipoprotein ◽

Density Lipoprotein ◽

Low Density ◽

Lipoprotein Receptor ◽

Dependent Manner ◽

Related Protein ◽

Low Density Lipoprotein Receptor ◽

Density Lipoprotein Receptor ◽

Lipoprotein Receptor Related Protein ◽

Pai 1

The low-density lipoprotein receptor-related protein (LRP) is a 600-kD scavenger receptor that binds a number of protein ligands with high affinity. Although some ligands do not compete with each other, binding of all is uniformly blocked by the 39-kD receptor-associated protein (RAP). RAP is normally found in the endoplasmic reticulum and seems to function as a chaperone for LRP. To identify the binding sites for RAP, lactoferrin, and plasminogen activator inhibitor-1 (PAI-1), a bacterial expression system has been developed to produce soluble LRP fragments spanning residues 783-1399. These residues overlap most of the CNBr fragment containing the second cluster of complement-type repeats (C). Solid phase binding assays show that 125I-RAP binds to fragments containing three successive complement-type repeats: C5-C7. PAI-1 and lactoferrin bind to the same fragments. A fragment containing C5-C7 also blocks uptake and degradation of 125I-RAP by fibroblasts in a concentration-dependent manner. Binding competition experiments show that RAP, PAI-1, and lactoferrin each inhibit the binding of the others, suggesting that at this site in LRP, RAP acts as a competitive, rather than an allosteric, inhibitor of PAI-1 and lactoferrin binding. © 1998 by The American Society of Hematology.

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Human α-defensin regulates smooth muscle cell contraction: a role for low-density lipoprotein receptor–related protein/α2-macroglobulin receptor

Blood ◽

10.1182/blood-2002-04-1080 ◽

2002 ◽

Vol 100 (12) ◽

pp. 4026-4032 ◽

Cited By ~ 61

Author(s):

Taher Nassar ◽

Sa'ed Akkawi ◽

Rachel Bar-Shavit ◽

Abdullah Haj-Yehia ◽

Khalil Bdeir ◽

...

Keyword(s):

Smooth Muscle ◽

Low Density Lipoprotein ◽

Density Lipoprotein ◽

Low Density ◽

Lipoprotein Receptor ◽

Related Protein ◽

Α2 Macroglobulin ◽

Density Lipoprotein Receptor ◽

Dose Dependent ◽

Lipoprotein Receptor Related Protein

We have previously identified α-defensin in association with medial smooth muscle cells (SMCs) in human coronary arteries. In the present paper we report that α-defensin, at concentrations below those found in pathological conditions, inhibits phenylephrine (PE)–induced contraction of rat aortic rings. Addition of 1 μM α-defensin increased the half-maximal effective concentration (EC50) of PE on denuded aortic rings from 32 to 630 nM. The effect of α-defensin was dose dependent and saturable, with a half-maximal effect at 1 μM. α-Defensin binds to human umbilical vein SMCs in a specific manner. The presence of 1 μM α-defensin inhibited the PE-mediated Ca++ mobilization in SMCs by more than 80%. The inhibitory effect of α-defensin on contraction of aortic rings and Ca++ mobilization was completely abolished by anti–low-density lipoprotein receptor–related protein/α2-macroglobulin receptor (LRP) antibodies as well as by the antagonist receptor-associated protein (RAP). α-Defensin binds directly to isolated LRP in a specific and dose-dependent manner; the binding was inhibited by RAP as well as by anti-LRP antibodies. α-Defensin is internalized by SMCs and interacts with 2 intracellular subtypes of protein kinase C (PKC) involved in muscle contraction, α and β. RAP and anti-LRP antibodies inhibited the binding and internalization of α-defensin by SMCs and its interaction with intracellular PKCs. These observations suggest that binding of α-defensin to LRP expressed in SMCs leads to its internalization; internalized α-defensin binds to PKC and inhibits its enzymatic activity, leading to decreased Ca++mobilization and SMC contraction in response to PE.

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