The plasminogen activation cascade system, directed by urokinase and the ur
okinase receptor, plays a key role in extracellular proteolysis during tiss
ue remodeling, To identify molecular interaction partners of these trigger
proteins on the cell, we combined covalent protein cross-linking with mass
spectrometry based methods for peptide mapping and primary structure analys
is of electrophoretically isolated protein conjugates, A specific tri-molec
ular complex was observed upon addition of pro-urokinase to human U937 cell
s. This complex included the urokinase receptor, pro-urokinase, and an unkn
own, high molecular weight urokinase receptor-associated protein. The trypt
ic peptide mixture derived from a cross-linked complex of pro-urokinase and
the latter protein was analyzed by nanoelectrospray tandem mass spectromet
ric sequencing. This analysis identified the novel protein as the human hom
ologue of a murine membrane-bound lectin with hitherto unknown function, Th
e human cDNA was cloned and sequenced. The protein, designated uPARAP, is a
member of the macrophage mannose receptor protein family and contains a pu
tative collagen-binding (fibronectin type II) domain in addition to 8 C-typ
e carbohydrate recognition domains. It proved capable of binding strongly t
o a single type of collagen, collagen V. This collagen binding reaction at
the exact site of plasminogen activation on the cell may lead to adhesive f
unctions as well as a contribution to cellular degradation of collagen matr
ices.