Faculty Collaboration Database

Residues essential for plasminogen binding by the cation-independent mannose 6-phosphate receptor. Biochemistry 2010 Jan 26;49(3):635-44

Date

12/24/2009

Pubmed ID

20028034

Pubmed Central ID

PMC2814163

DOI

10.1021/bi901779p

Scopus ID

2-s2.0-74949084342 (requires institutional sign-in at Scopus site)   11 Citations

Abstract

The 300 kDa cation-independent mannose 6-phosphate receptor (CI-MPR) is a multifunctional protein that binds diverse intracellular and extracellular ligands with high affinity. The CI-MPR is a receptor for plasminogen, and this interaction can be inhibited by lysine analogues. To characterize the molecular basis for this interaction, surface plasmon resonance (SPR) analyses were performed using truncated forms of the CI-MPR and plasminogen. The results show that the N-terminal region of the CI-MPR containing domains 1 and 2, but not domain 1 alone, of the receptor's 15-domain extracytoplasmic region binds plasminogen (K(d) = 5 +/- 1 nM) with an affinity similar to that of the full-length receptor (K(d) = 20 +/- 6 nM). In addition to its C-terminal serine protease domain, plasminogen contains lysine binding sites (LBS), which are located within each of its five kringle domains, except kringle 3. We show that kringles 1-4, but not kringles 1-3, bind the CI-MPR, indicating an essential role for the LBS in kringle 4 of plasminogen. To identify the lysine residue(s) of the CI-MPR that serve(s) as an essential determinant for recognition by the LBS of plasminogen, site-directed mutagenesis studies were carried out using a construct encoding the N-terminal three domains of the CI-MPR (Dom1-3His) which contains both a mannose 6-phosphate (Man-6-P) and plasminogen binding site. The results demonstrate two lysine residues (Lys53 located in domain 1 and Lys125 located in the loop connecting domains 1 and 2) of the CI-MPR are key determinants for plasminogen binding but are not required for Man-6-P binding.

Author List

Bohnsack RN, Patel M, Olson LJ, Twining SS, Dahms NM

Authors

Richard N. Bohnsack Research Scientist I in the Biochemistry department at Medical College of Wisconsin
Nancy M. Dahms PhD Professor in the Biochemistry department at Medical College of Wisconsin
Linda J. Olson PhD Assistant Professor in the Pharmacology and Toxicology department at Medical College of Wisconsin
Sally S. Twining PhD Assistant Dean, Professor in the Biochemistry department at Medical College of Wisconsin

MESH terms used to index this publication - Major topics in bold

Animals
Binding Sites
Cations
Cattle
Humans
Kinetics
Kringles
Ligands
Lysine
Plasminogen
Receptor, IGF Type 2
Substrate Specificity
Surface Plasmon Resonance