PMID- 8798468
OWN - NLM
STAT- MEDLINE
DCOM- 19961107
LR  - 20210210
IS  - 0021-9258 (Print)
IS  - 0021-9258 (Linking)
VI  - 271
IP  - 37
DP  - 1996 Sep 13
TI  - Domain interplay in the urokinase receptor. Requirement for the third domain in 
      high affinity ligand binding and demonstration of ligand contact sites in 
      distinct receptor domains.
PG  - 22885-94
AB  - The urokinase plasminogen activator receptor (uPAR) is a membrane protein 
      comprised of three extracellular domains. In order to study the importance of 
      this domain organization in the ligand-binding process of the receptor we 
      subjected a recombinant, soluble uPAR (suPAR) to specific proteolytic cleavages 
      leading to liberation of single domains. Treatment of the receptor with pepsin 
      resulted in cleavage between residues 183 and 184, thus separating the third 
      domain (D3) from the rest of the molecule, which was left as an intact fragment 
      (D(1 + 2)). D(1 + 2) proved capable of ligand binding as shown by chemical 
      cross-linking, but quantitative binding/competition studies showed that the 
      apparent ligand affinity was 100- to 1000-fold lower than that of the intact 
      suPAR. This loss of affinity was comparable with the loss found after cleavage 
      between the first domain (D1) and D(2 + 3), using chymotrypsin. This result shows 
      that in addition to D1, which has an established function in ligand binding 
      (Behrendt, N., Ploug, M., Patthy, L., Houen, G., Blasi, F., and Dano, K. (1991) 
      J. Biol. Chem. 266, 7842-7847), D3 has an important role in governing a high 
      affinity in the intact receptor. Real-time biomolecular interaction analysis 
      revealed that the decrease in affinity was caused mostly by an increased 
      dissociation rate of the ligand complex of D(1 + 2). Zero length cross-linking, 
      using carbodiimide-induced, direct condensation, was used to identify regions 
      within suPAR engaged in molecular ligand contact. The purified suPAR was 
      cross-linked to the radiolabeled amino-terminal fragment (ATF) of urokinase, 
      followed by cleavage with chymotrypsin. In accordance with the cleavage pattern 
      found for the uncomplexed receptor, this treatment led to cleavage between D1 and 
      D(2 + 3). Analysis of the radiolabeled fragments revealed the expected ligand 
      labeling of D1 but a clear labeling of D(2 + 3) was also found, indicating that 
      this part of the molecule is also situated in close contact with ATF in the 
      receptor-ligand complex. The latter contact site may contribute to the role of 
      molecular regions outside D1 in high affinity binding.
FAU - Behrendt, N
AU  - Behrendt N
AD  - Finsen Laboratory, Rigshospitalet, Strandboulevarden 49, Building 7. 2, DK-2100 
      Copenhagen O, Denmark.
FAU - Ronne, E
AU  - Ronne E
FAU - Dano, K
AU  - Dano K
LA  - eng
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
PL  - United States
TA  - J Biol Chem
JT  - The Journal of biological chemistry
JID - 2985121R
RN  - 0 (Receptors, Cell Surface)
RN  - 0 (Receptors, Urokinase Plasminogen Activator)
RN  - EC 3.4.21.73 (Urokinase-Type Plasminogen Activator)
RN  - EC 3.4.23.1 (Pepsin A)
SB  - IM
MH  - Amino Acid Sequence
MH  - Animals
MH  - Binding Sites
MH  - Binding, Competitive
MH  - Cell Line
MH  - Chromatography, Gel
MH  - Chromatography, High Pressure Liquid
MH  - Cricetinae
MH  - Cricetulus
MH  - Electrophoresis, Polyacrylamide Gel
MH  - Female
MH  - Kinetics
MH  - Molecular Sequence Data
MH  - Ovary/metabolism
MH  - Pepsin A/metabolism
MH  - Protein Conformation
MH  - Receptors, Cell Surface/*metabolism
MH  - Receptors, Urokinase Plasminogen Activator
MH  - Structure-Activity Relationship
MH  - Urokinase-Type Plasminogen Activator/metabolism
EDAT- 1996/09/13 00:00
MHDA- 1996/09/13 00:01
CRDT- 1996/09/13 00:00
PHST- 1996/09/13 00:00 [pubmed]
PHST- 1996/09/13 00:01 [medline]
PHST- 1996/09/13 00:00 [entrez]
AID - S0021-9258(19)61834-X [pii]
AID - 10.1074/jbc.271.37.22885 [doi]
PST - ppublish
SO  - J Biol Chem. 1996 Sep 13;271(37):22885-94. doi: 10.1074/jbc.271.37.22885.