S U M M A R YRecent studies suggest important functions for laminin-8 (Ln-8; ␣ 4 ␤ 1 ␥ 1) in vascular and blood cell biology, but its distribution in human tissues has remained elusive. We have raised a monoclonal antibody (MAb) FC10, and by enzyme-linked immunoassay (EIA) and Western blotting techniques we show that it recognizes the human Ln ␣ 4-chain. Immunoreactivity for the Ln ␣ 4-chain was localized in tissues of mesodermal origin, such as basement membranes (BMs) of endothelia, adipocytes, and skeletal, smooth, and cardiac muscle cells. In addition, the Ln ␣ 4-chain was found in regions of some epithelial BMs, including epidermis, salivary glands, pancreas, esophageal and gastric glands, intestinal crypts, and some renal medullary tubules. Developmental differences in the distribution of Ln ␣ 4-chain were detected in skeletal muscle, walls of vessels, and intestinal crypts. Ln ␣ 4-and Ln ␣ 2-chains co-localized in BMs of fetal skeletal muscle cells and in some epithelial BMs, e.g., in gastric glands and acini of pancreas. Cultured human pulmonary artery endothelial (HPAE) cells produced Ln ␣ 4-chain as M r 180,000 and 200,000 doublet and rapidly deposited it to the growth substratum. In cell-free extracellular matrices of human kidney and lung, Ln ␣ 4-chain was found as M r 180,000 protein.

Laminins, a growing family of large heterotrimeric proteins with cell adhesive and signaling properties, are major components of vascular and other basement membranes. Expression, recognition, and use of laminin isoforms by leukocytes are poorly understood. In monoblastic THP-1 cells, transcripts for laminin γ1-, β1-, and α4-chains were detected by RT-PCR. Following immunoaffinity purification on a laminin β1 Ab-Sepharose column, laminin β1- (220 kDa), γ1- (200 kDa), and α4- (180/200 kDa) chains were detected by Western blotting in THP-1 cells and in two other monoblastic cell lines, U-937 and Mono Mac 6. After cell permeabilization, a mAb to laminin γ1-chain reacted with practically all blood monocytes by immunofluorescence flow cytometry, and laminin-8 (α4β1γ1) could be isolated also from these cells. Monoblastic JOSK-I cells adhered constitutively to immobilized recombinant laminin-8, less than to laminin-10/11 (α5β1γ1/α5β2γ1) but to a higher level than to laminin-1 (α1β1γ1). Compared with the other laminin isoforms, adhesion to laminin-8 was preferentially mediated by α6β1 and β2 integrins. Laminin-8 and, to a lower extent, laminin-1 promoted spontaneous and chemokine-induced migration of blood monocytes, whereas laminin-10/11 was inhibitory. Altogether, the results indicate that leukocytes, as other cell types, are able to synthesize complete laminin molecules. Expression, recognition, and use of laminin-8 by leukocytes suggest a major role of this laminin isoform in leukocyte physiology.

Photoaffinity cross-linking with [azidobenzoyl-d-Lys6]GnRH leads to irreversible activation of the gonadotropin-releasing hormone (GnRH) receptor. In order to localize the cross-linking site, the disulfide bridge structure was initially probed by mutagenesis. A consistent pattern of changes in the ability of GnRH to stimulate signal transduction after Ser substitutions of extracellularly located Cys residues indicated that Cys14 in the N-terminal domain is connected to Cys200 in the second extracellular loop, while Cys196 in this loop is connected to the highly conserved Cys114 at the extracellular end of transmembrane helix 3. Protein chemical analysis of radioactive fragments of cross-linked GnRH receptor following deglycosylation and enzymatic digest with endoproteinase Glu-C and trypsin before and after introduction or elimination of potential protease cleavage sites indicated that 125I[azidobenzoyl-d-Lys6]GnRH cross-links to a segment comprising residues 12-18 of the N-terminal domain. The existence of the Cys114-Cys196 bridge was directly confirmed as a labeled fragment, including that Cys114 was resolvable only under reducing conditions. The observation that the cross-linked N-terminal enzymatic fragments had identical apparent size under non-reducing conditions shows that the cross-linking reaction disconnected the disulfide bridge between Cys14 and Cys200 and indicates that Cys14 is probably the residue involved in cross-linking of the ligand. It is concluded that covalent tethering of GnRH through a photoreactive side chain located at position 6 in the middle of the peptide leads to continued activation of the receptor presumably through covalent binding to Cys14 in the N-terminal domain of the receptor.

Promastigotes of L. aethiopica express an ectokinase activity preferring histone V-S as substrate. A soluble kinase activity utilizing protamine and histone V-S, as well as a particulate fraction associated kinase activity preferring protamine are also expressed. The soluble histone kinase activity, but not the ectokinase, was expressed at a higher level in cells from late phases of growth, as compared to early log phase cultures. Transformation of L. aethiopica to an amastigote-like stage, resulted in almost complete loss of the kinase activities, with retained viability of the cells. Formycin-ATP only weakly inhibited the kinases while effectively inhibiting cell growth and thymidine incorporation. Staurosporin efficiently blocked the kinase activities and cell growth without affecting thymidine incorporation.