Cell surface targeting of pregnancy-associated plasma protein A proteolytic activity. Reversible adhesion is mediated by two neighboring short consensus repeats

TY - JOUR

T1 - Cell surface targeting of pregnancy-associated plasma protein A proteolytic activity. Reversible adhesion is mediated by two neighboring short consensus repeats

AU - Laursen, Lisbeth S

AU - Overgaard, Michael T

AU - Weyer, Kathrin

AU - Boldt, Henning B

AU - Ebbesen, Peter

AU - Christiansen, Michael

AU - Sottrup-Jensen, Lars

AU - Giudice, Linda C

AU - Oxvig, Claus

PY - 2002/12/6

Y1 - 2002/12/6

N2 - The activities of insulin-like growth factor (IGF)-I and -II are regulated by IGF-binding proteins (IGFBPs). Cleavage of IGFBP-4 by the metalloproteinase pregnancy-associated plasma protein-A (PAPP-A) causes release of bound IGF and has been established in several biological systems including the human reproductive system. Using flow cytometry, we first demonstrate that PAPP-A reversibly binds to the cell surface of several cell types analyzed. Heparin and heparan sulfate, but not dermatan or chondroitin sulfate, effectively compete for PAPP-A surface binding, and because incubation of cells with heparinase abrogated PAPP-A adhesion, binding is probably mediated by a cell surface heparan sulfate proteoglycan. Furthermore, the proteolytic activity of PAPP-A is preserved while bound to cells, suggesting that adhesion functions to target its activity to the vicinity of the IGF receptor, decreasing the probability that released IGF is captured by another IGFBP molecule before receptor binding. This mechanism potentially functions in both autocrine and paracrine regulation, as PAPP-A need not be synthesized in a cell to which it adheres. A truncated PAPP-A variant without the five short consensus repeats in the C-terminal third of the 1547-residue PAPP-A subunit, lacked surface binding. We also show that PAPP-A2, a recently discovered IGFBP-5 proteinase with homology to PAPP-A, does not bind cells. This finding allowed further mapping of the PAPP-A adhesion site to short consensus repeat modules 3 and 4 by the expression and analysis of nine PAPP-A/PAPP-A2 chimeras. Interestingly, the proteolytically inactive, disulfide-bound complex of PAPP-A and the proform of eosinophil major basic protein (proMBP), PAPP-A.proMBP, shows only weak surface binding, probably because the adhesion site of PAPP-A is occupied by heparan sulfate, known to be covalently bound to proMBP. This hypothesis was further substantiated by demonstrating that heparinase treatment of PAPP-A.proMBP restores surface binding. We finally propose a model in which IGF bioactivity is regulated by reversible cell surface binding of PAPP-A, which in turn is regulated by proMBP.

AB - The activities of insulin-like growth factor (IGF)-I and -II are regulated by IGF-binding proteins (IGFBPs). Cleavage of IGFBP-4 by the metalloproteinase pregnancy-associated plasma protein-A (PAPP-A) causes release of bound IGF and has been established in several biological systems including the human reproductive system. Using flow cytometry, we first demonstrate that PAPP-A reversibly binds to the cell surface of several cell types analyzed. Heparin and heparan sulfate, but not dermatan or chondroitin sulfate, effectively compete for PAPP-A surface binding, and because incubation of cells with heparinase abrogated PAPP-A adhesion, binding is probably mediated by a cell surface heparan sulfate proteoglycan. Furthermore, the proteolytic activity of PAPP-A is preserved while bound to cells, suggesting that adhesion functions to target its activity to the vicinity of the IGF receptor, decreasing the probability that released IGF is captured by another IGFBP molecule before receptor binding. This mechanism potentially functions in both autocrine and paracrine regulation, as PAPP-A need not be synthesized in a cell to which it adheres. A truncated PAPP-A variant without the five short consensus repeats in the C-terminal third of the 1547-residue PAPP-A subunit, lacked surface binding. We also show that PAPP-A2, a recently discovered IGFBP-5 proteinase with homology to PAPP-A, does not bind cells. This finding allowed further mapping of the PAPP-A adhesion site to short consensus repeat modules 3 and 4 by the expression and analysis of nine PAPP-A/PAPP-A2 chimeras. Interestingly, the proteolytically inactive, disulfide-bound complex of PAPP-A and the proform of eosinophil major basic protein (proMBP), PAPP-A.proMBP, shows only weak surface binding, probably because the adhesion site of PAPP-A is occupied by heparan sulfate, known to be covalently bound to proMBP. This hypothesis was further substantiated by demonstrating that heparinase treatment of PAPP-A.proMBP restores surface binding. We finally propose a model in which IGF bioactivity is regulated by reversible cell surface binding of PAPP-A, which in turn is regulated by proMBP.

KW - Binding Sites

KW - Blood Proteins

KW - Blotting, Western

KW - Cell Adhesion

KW - Cell Line

KW - Cell Membrane

KW - Cysteine

KW - DNA, Complementary

KW - Dose-Response Relationship, Drug

KW - Enzyme-Linked Immunosorbent Assay

KW - Eosinophil Granule Proteins

KW - Flow Cytometry

KW - Glycosaminoglycans

KW - Heparin

KW - Heparitin Sulfate

KW - Humans

KW - Insulin-Like Growth Factor I

KW - Models, Biological

KW - Plasmids

KW - Pregnancy-Associated Plasma Protein-A

KW - Protein Binding

KW - Protein Structure, Tertiary

KW - Ribonucleases

KW - Transfection

U2 - 10.1074/jbc.M209155200

DO - 10.1074/jbc.M209155200

M3 - Journal article

C2 - 12370176

SN - 0021-9258

VL - 277

SP - 47225

EP - 47234

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

IS - 49

ER -