Sites within the 39-kDa protein important for regulating ligand binding to the low-density lipoprotein receptor-related protein

I. Warshawsky, Guojun D Bu, A. L. Schwartz

Research output: Contribution to journalArticle

33 Citations (Scopus)

Abstract

A 39-kDa protein copurifies with the low-density lipoprotein receptor- related protein/α2-macroglobulin receptor (LRP) and inhibits the binding and/or cellular uptake of ligands by this receptor. We recently utilized glutathione S-transferase (GST)-39-kDa fusion protein constructs to demonstrate that constructs encoding amino-terminal residues 1-114 and carboxy-terminal residues 115-319 of the 39-kDa protein independently bind to purified LRP and to LRP on hepatoma cells with similar affinities as the full-length GST-39-kDa protein (K(d) ~ 8-10 nM). These regions, however, inhibit ligand binding to LRP differently: GST/1-114 inhibits both tissue- type plasminogen activator (t-PA) and α2-macroglobulin-methylamine (α2M*) binding whereas GST/115-319 only potently inhibits t-PA binding. Four domains, containing residues 18-24 and 100-107 within amino-terminal constructs and residues 200-225 and 311-319 within carboxy-terminal constructs, are required for inhibition of ligand binding. In the present study, we generated additional 39-kDa protein constructs to precisely define residues within each domain required for inhibition of t-PA and α2M* binding to LRP. The potential importance of these residues in mediating direct binding both to purified LRP and to LRP on hepatoma cells was examined. Within amino-terminal residues 1-114, alanine 103 and leucine 104 are required for inhibition of t-PA and α2M* binding. These residues, however, are not required for binding either to purified LRP or to LRP on hepatoma cells. Within domain 18-24, arginine 21 is required for inhibition of t-PA and α2M* binding as well as for the direct binding of amino- terminal constructs to LRP. Within carboxy-terminal domains 200-225 and 311- 319, leucine 222 and leucine 319 are both required for inhibition of t-PA binding. Deletion of leucine 319 changes the ligand specificity from inhibition of t-PA binding to inhibition of α2M* binding. Thus, leucine 319 is not required for direct binding to LRP whereas leucine 222 is required for high-affinity binding to LRP.

Original languageEnglish (US)
Pages (from-to)3404-3415
Number of pages12
JournalBiochemistry
Volume34
Issue number10
DOIs
StatePublished - 1995
Externally publishedYes

Fingerprint

LDL-Receptor Related Proteins
Macroglobulins
LDL Receptors
Plasminogen Activators
Leucine
Ligands
Glutathione Transferase
Hepatocellular Carcinoma
Proteins
Low Density Lipoprotein Receptor-Related Protein-2
Tissue Plasminogen Activator
Alanine
Arginine
Fusion reactions
methylamine

ASJC Scopus subject areas

  • Biochemistry

Cite this

Sites within the 39-kDa protein important for regulating ligand binding to the low-density lipoprotein receptor-related protein. / Warshawsky, I.; Bu, Guojun D; Schwartz, A. L.

In: Biochemistry, Vol. 34, No. 10, 1995, p. 3404-3415.

Research output: Contribution to journalArticle

@article{514ccbe4009340d9a2cb950d7eb85e32,
title = "Sites within the 39-kDa protein important for regulating ligand binding to the low-density lipoprotein receptor-related protein",
abstract = "A 39-kDa protein copurifies with the low-density lipoprotein receptor- related protein/α2-macroglobulin receptor (LRP) and inhibits the binding and/or cellular uptake of ligands by this receptor. We recently utilized glutathione S-transferase (GST)-39-kDa fusion protein constructs to demonstrate that constructs encoding amino-terminal residues 1-114 and carboxy-terminal residues 115-319 of the 39-kDa protein independently bind to purified LRP and to LRP on hepatoma cells with similar affinities as the full-length GST-39-kDa protein (K(d) ~ 8-10 nM). These regions, however, inhibit ligand binding to LRP differently: GST/1-114 inhibits both tissue- type plasminogen activator (t-PA) and α2-macroglobulin-methylamine (α2M*) binding whereas GST/115-319 only potently inhibits t-PA binding. Four domains, containing residues 18-24 and 100-107 within amino-terminal constructs and residues 200-225 and 311-319 within carboxy-terminal constructs, are required for inhibition of ligand binding. In the present study, we generated additional 39-kDa protein constructs to precisely define residues within each domain required for inhibition of t-PA and α2M* binding to LRP. The potential importance of these residues in mediating direct binding both to purified LRP and to LRP on hepatoma cells was examined. Within amino-terminal residues 1-114, alanine 103 and leucine 104 are required for inhibition of t-PA and α2M* binding. These residues, however, are not required for binding either to purified LRP or to LRP on hepatoma cells. Within domain 18-24, arginine 21 is required for inhibition of t-PA and α2M* binding as well as for the direct binding of amino- terminal constructs to LRP. Within carboxy-terminal domains 200-225 and 311- 319, leucine 222 and leucine 319 are both required for inhibition of t-PA binding. Deletion of leucine 319 changes the ligand specificity from inhibition of t-PA binding to inhibition of α2M* binding. Thus, leucine 319 is not required for direct binding to LRP whereas leucine 222 is required for high-affinity binding to LRP.",
author = "I. Warshawsky and Bu, {Guojun D} and Schwartz, {A. L.}",
year = "1995",
doi = "10.1021/bi00010a032",
language = "English (US)",
volume = "34",
pages = "3404--3415",
journal = "Biochemistry",
issn = "0006-2960",
publisher = "American Chemical Society",
number = "10",

}

TY - JOUR

T1 - Sites within the 39-kDa protein important for regulating ligand binding to the low-density lipoprotein receptor-related protein

AU - Warshawsky, I.

AU - Bu, Guojun D

AU - Schwartz, A. L.

PY - 1995

Y1 - 1995

N2 - A 39-kDa protein copurifies with the low-density lipoprotein receptor- related protein/α2-macroglobulin receptor (LRP) and inhibits the binding and/or cellular uptake of ligands by this receptor. We recently utilized glutathione S-transferase (GST)-39-kDa fusion protein constructs to demonstrate that constructs encoding amino-terminal residues 1-114 and carboxy-terminal residues 115-319 of the 39-kDa protein independently bind to purified LRP and to LRP on hepatoma cells with similar affinities as the full-length GST-39-kDa protein (K(d) ~ 8-10 nM). These regions, however, inhibit ligand binding to LRP differently: GST/1-114 inhibits both tissue- type plasminogen activator (t-PA) and α2-macroglobulin-methylamine (α2M*) binding whereas GST/115-319 only potently inhibits t-PA binding. Four domains, containing residues 18-24 and 100-107 within amino-terminal constructs and residues 200-225 and 311-319 within carboxy-terminal constructs, are required for inhibition of ligand binding. In the present study, we generated additional 39-kDa protein constructs to precisely define residues within each domain required for inhibition of t-PA and α2M* binding to LRP. The potential importance of these residues in mediating direct binding both to purified LRP and to LRP on hepatoma cells was examined. Within amino-terminal residues 1-114, alanine 103 and leucine 104 are required for inhibition of t-PA and α2M* binding. These residues, however, are not required for binding either to purified LRP or to LRP on hepatoma cells. Within domain 18-24, arginine 21 is required for inhibition of t-PA and α2M* binding as well as for the direct binding of amino- terminal constructs to LRP. Within carboxy-terminal domains 200-225 and 311- 319, leucine 222 and leucine 319 are both required for inhibition of t-PA binding. Deletion of leucine 319 changes the ligand specificity from inhibition of t-PA binding to inhibition of α2M* binding. Thus, leucine 319 is not required for direct binding to LRP whereas leucine 222 is required for high-affinity binding to LRP.

AB - A 39-kDa protein copurifies with the low-density lipoprotein receptor- related protein/α2-macroglobulin receptor (LRP) and inhibits the binding and/or cellular uptake of ligands by this receptor. We recently utilized glutathione S-transferase (GST)-39-kDa fusion protein constructs to demonstrate that constructs encoding amino-terminal residues 1-114 and carboxy-terminal residues 115-319 of the 39-kDa protein independently bind to purified LRP and to LRP on hepatoma cells with similar affinities as the full-length GST-39-kDa protein (K(d) ~ 8-10 nM). These regions, however, inhibit ligand binding to LRP differently: GST/1-114 inhibits both tissue- type plasminogen activator (t-PA) and α2-macroglobulin-methylamine (α2M*) binding whereas GST/115-319 only potently inhibits t-PA binding. Four domains, containing residues 18-24 and 100-107 within amino-terminal constructs and residues 200-225 and 311-319 within carboxy-terminal constructs, are required for inhibition of ligand binding. In the present study, we generated additional 39-kDa protein constructs to precisely define residues within each domain required for inhibition of t-PA and α2M* binding to LRP. The potential importance of these residues in mediating direct binding both to purified LRP and to LRP on hepatoma cells was examined. Within amino-terminal residues 1-114, alanine 103 and leucine 104 are required for inhibition of t-PA and α2M* binding. These residues, however, are not required for binding either to purified LRP or to LRP on hepatoma cells. Within domain 18-24, arginine 21 is required for inhibition of t-PA and α2M* binding as well as for the direct binding of amino- terminal constructs to LRP. Within carboxy-terminal domains 200-225 and 311- 319, leucine 222 and leucine 319 are both required for inhibition of t-PA binding. Deletion of leucine 319 changes the ligand specificity from inhibition of t-PA binding to inhibition of α2M* binding. Thus, leucine 319 is not required for direct binding to LRP whereas leucine 222 is required for high-affinity binding to LRP.

UR - http://www.scopus.com/inward/record.url?scp=0028933638&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0028933638&partnerID=8YFLogxK

U2 - 10.1021/bi00010a032

DO - 10.1021/bi00010a032

M3 - Article

C2 - 7533537

AN - SCOPUS:0028933638

VL - 34

SP - 3404

EP - 3415

JO - Biochemistry

JF - Biochemistry

SN - 0006-2960

IS - 10

ER -