Residues essential for plasminogen binding by the cation-independent mannose 6-phosphate receptor.
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.
Bohnsack
Richard N
RN
Department of Biochemistry, Medical College of Wisconsin, Milwaukee, Wisconsin 53226, USA.
Patel
Manish
M
Olson
Linda J
LJ
Twining
Sally S
SS
Dahms
Nancy M
NM
D000818:Animals; D001665:Binding Sites; D002412:Cations / Q000737:chemistry; D002417:Cattle; D006801:Humans; D007700:Kinetics; D018082:Kringles; D008024:Ligands; D008239:Lysine / Q000235:genetics / Q000378:metabolism; D010958:Plasminogen / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017527:Receptor, IGF Type 2 / Q000737:chemistry* / Q000378:metabolism*; D013379:Substrate Specificity; D020349:Surface Plasmon Resonance
D002412:Cations; D008024:Ligands; D017527:Receptor, IGF Type 2; D010958:Plasminogen; D008239:Lysine