Subcellular targeting of multiligand-binding protein gC1qR.

gC1q receptor, a protein originally described as the cell surface receptor for the globular heads of complement factor C1q, has been found to bind human H-kininogen with high affinity and specificity. Therefore, gC1qR has been considered candidate kininogen docking site on the surfaces of platelets, neutrophils and endothelial cells. Recent work demonstrating that gC1qR is an intracellular protein that is tightly associated with mitochondria rather than targeted to the cell surface has challenged this view.

Mapping of the discontinuous H-kininogen binding site of plasma prekallikrein. Evidence for a critical role of apple domain-2.

Plasma prekallikrein, a zymogen of the contact phase system, circulates in plasma as heterodimeric complex with H-kininogen. The binding is mediated by the prekallikrein heavy chain consisting of four apple domains, A1 to A4, to which H-kininogen binds with high specificity and affinity (K(D) = 1.2 x 10(-8) M).

The multiligand-binding protein gC1qR, putative C1q receptor, is a mitochondrial protein.

A protein of 33 kDa (p33) that tightly binds to the globular domains of the first complement component, C1q, is thought to serve as the major C1q receptor (gC1qR) on B cells, neutrophils, and mast cells. However, the cellular routing and the subcellular localization of p33/gC1qR are unknown. We have performed confocal laser-scanning microscopy and found that p33/gC1qR is present in intracellular compartments, where it colocalizes with the mitochondrial marker protein, pyruvate dehydrogenase.

Kininogen binding protein p33/gC1qR is localized in the vesicular fraction of endothelial cells.

The endothelial protein p33/gC1qR is thought to mediate the assembly of components of the kinin-forming and complement-activating pathways on the surface of cardiovascular cells. FACS analysis of intact human umbilical vein endothelial cells using specific antibodies to p33 revealed a minor fluorescence on the cell surface whereas permeabilized cells showed a bright fluorescence indicative of an intracellular localization of p33. Immunostaining of fixed cells confirmed the predominant intracellular localization of p33.

Ligand-induced phosphorylation/dephosphorylation of the endogenous bradykinin B2 receptor from human fibroblasts.

We have studied the ligand-induced phosphorylation/dephosphorylation of the bradykinin B2 receptor endogenously expressed in human HF-15 fibroblasts. An antiserum (AS346) to a synthetic peptide (CRS36), derived from the extreme carboxyl terminus of the human B2 receptor, precipitated the receptor from solubilized membranes of HF-15 cells that had been labeled with [32P]orthophosphate. A low basal level of B2 receptor phosphorylation was found in the absence of a ligand.

Mapping of the discontinuous kininogen binding site of prekallikrein. A distal binding segment is located in the heavy chain domain A4.

Prekallikrein, the precursor to the serine proteinase kallikrein, circulates in plasma in an equimolar complex with H-kininogen. The binding to H-kininogen is mediated by the kallikrein heavy chain consisting of four "apple" domains, A1-A4, which attaches to H-kininogen with high specificity and affinity (KD = 83 nM). At least two distinct portions of the kallikrein heavy chain form this H-kininogen binding site: a proximal segment located in the NH2-terminal fragment of the heavy chain encompassing A1, and distal segment(s) located in COOH-terminal fragment spanning domains A2-A4.

Isolation and characterization of the kininogen-binding protein p33 from endothelial cells. Identity with the gC1q receptor.

Kininogens, the precursor proteins of the vasoactive kinins, bind specifically, reversibly, and saturably to platelets, neutrophils, and endothelial cells. Two domains of the kininogens expose major cell binding sites: domain D3 that is shared by H- and L-kininogen and domain D5H that is exclusively present in H-kininogen. Previously we have mapped the kininogen cell binding sites to 27 residues of D3 ("LDC27") and 20 residues of D5H ("HKH20"", respectively (Herwald, H.

Extracellular domains of the bradykinin B2 receptor involved in ligand binding and agonist sensing defined by anti-peptide antibodies.

Many of the physiological functions of bradykinin are mediated via the B2 receptor. Little is known about binding sites for bradykinin on the receptor. Therefore, antisera against peptides derived from the putative extracellular domains of the B2 receptor were raised.