We report the immunoaffinity isolation of bradykinin B2 receptors in a tyrosine-phosphorylated state from WI-38 human lung fibroblasts. We generated six monoclonal antibodies directed against B2 bradykinin receptor biologic activity mediating prostaglandin E2 production in WI- Bradykinin (BK), Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg, is a multifunctional mediator that aids in maintaining tissue homeostasis and plays a key role in events accompanying inflammation. BK is proteolytically generated from kininogen precursors in circulation and interstitial tissue fluids; its local concentration is rapidly increased upon tissue injury. BK receptors belonging to the guanine nucleotide-binding protein (G-protein)-coupled family trigger neuronal transmission of pain or irritant messages, enhancement of vascular permeability, and altered vasomotor tone that are prominent features of the inflammatory process. BK action on these same receptors also regulates mitogenesis and protein production in fibroblasts, initiating repair steps that restore normal tissue form and function following injury (1-4).We (1) previously demonstrated that IMR-90 human lung fibroblasts sequentially mobilize BK B2 subtype receptors of Kd = 2.5-5 nM and 44 nM that mediate prostaglandin E2 (PGE2) production, upon progression through their finite life span in vitro. Further information is needed at the cellular and molecular level to elucidate the nature of BK B2 receptors and the means by which cells can express diverse receptor forms. Although recent cloning of two BK receptors has disclosed their primary sequences (5-7), the molecular structure of the protein as expressed in intact cells remains uncharacterized. We have generated monoclonal antibodies (mAbs) that distinguish the BK receptors of Kd = 5 nM and 44 nM in human lung fibroblasts and have used them to isolate the receptor protein and to demonstrate that it containsThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. phosphorylated tyrosine, which may serve a regulatory function. We also show that tyrosine kinase activity is essential for BK-mediated PGE2 production. These mAbs will allow us to explore the role of BK receptors in the growth and development of fibroblasts and in these cells' participation in onset and/or propagation of the injury/inflammation response. 4°C was covalently crosslinked to receptors with 0.4 mM disuccinyl suberimidate (Pierce) at 4°C for 20 min. After quenching with 150 mM Tris (pH 8), cells were solubilized 2.5 min in 400 ,ul of 1% Triton X-100 or 12 mM 3-[(3-cholamidopropyl)dimethylammonio-1-propane sulfonate (CHAPS)/ 0.5% digitonin (CD) in 0.15 M NaCl/15 mM Tris Cl, pH 7.4, containing protease inhibitors (100 ,uM phenylmethanesulfonyl fluoride, 16.8 uM leupeptin, chymostatin at 4 ,ug/ml, 5.8 ,tM pepstatin, 6.6 ;LM antipain, 0.08 trypsin inhibitor unit of aprotinin per ml, 10 mM benzamidine, soybe...

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Coated and noncoated vesicles participate in cellular protein transport. Both acetylcholine receptors (AChR) and acetylcholinesterase (AChE) are transported via coated vesicles, some of which accumulate beneath the neuromuscular synapse where AChRs cluster. To investigate the mechanisms by which these proteins are transported during postsynaptic remodeling, we purified coated vesicles from the bovine brain via column chromatography (Sephacryl S-1000) and raised monoclonal antibodies to epitopes of the vesicular membranes enriched in AChE. We assayed for AChE (coated vesicle enriched), hexosaminidase (lysosomal contaminants), NADH cytochrome C reductase (mitochondrial containing), and protein and demonstrated electron microscopically using negative staining that the vesicular fraction contained 95% pure coated vesicles. We then injected coated vesicle fractions and the fractions from which the coat was removed intraperitoneally into mice and obtained three monoclonal antibodies: C-33, C-172, and F-22. On immunoblots of purified vesicles and cultured skeletal muscle, mAb C-33 stained a 180 Kd band and mAb C-172 stained a 100 kd band. MAb F-22 stained 50 kd and 55 kd bands and was not characterized further. Immunofluorescent microscopy with C-33 and C-172 revealed punctate fluorescence whose distribution depends upon the stage of myotube development. Four days after plating, myotubes showed punctate fluorescence throughout the myotube, whereas those stained 8 days after plating showed a punctate perinuclear distribution. Myotubes innervated by ciliary neurons show punctate fluorescence limited to the nuclear periphery and most concentrated around nuclei which line up beneath neuronal processes. This differential vesicular distribution, observed during myotube differentiation and innervation, suggests that these vesicles participate in vesicular membrane traffic.

The serious and growing impact of the neurodegenerative disorder Alzheimer's disease (AD) as an individual and societal burden raises a number of key questions: Can a blanket test for Alzheimer's disease be devised forecasting long-term risk for acquiring this disorder? Can a unified therapy be devised to forestall the development of AD as well as improve the lot of present sufferers? Inflammatory and oxidative stresses are associated with enhanced risk for AD. Can an AD molecular signature be identified in signaling pathways for communication within and among cells during inflammatory and oxidative stress, suggesting possible biomarkers and therapeutic avenues? We postulated a unique molecular signature of dysfunctional activity profiles in AD-relevant signaling pathways in peripheral tissues, based on a gain of function in G-protein-coupled bradykinin B2 receptor (BKB2R) inflammatory stress signaling in skin fibroblasts from AD patients that results in tau protein Ser hyperphosphorylation. Such a signaling profile, routed through both phosphorylation and proteolytic cascades activated by inflammatory and oxidative stresses in highly penetrant familial monogenic forms of AD, could be informative for pathogenesis of the complex multigenic sporadic form of AD. Comparing stimulus-specific cascades of signal transduction revealed a striking diversity of molecular signaling profiles in AD human skin fibroblasts that express endogenous levels of mutant presenilins PS-1 or PS-2 or the Trisomy 21 proteome. AD fibroblasts bearing the PS-1 M146L mutation associated with highly aggressive AD displayed persistent BKB2R signaling plus decreased ERK activation by BK, correctible by gamma-secretase inhibitor Compound E. Lack of these effects in the homologous PS-2 mutant cells indicates specificity of presenilin gamma-secretase catalytic components in BK signaling biology directed toward MAPK activation. Oxidative stress revealed a JNK-dependent survival pathway in normal fibroblasts lost in PS-1 M146L fibroblasts. Complex molecular profiles of signaling dysfunction in the most putatively straightforward human cellular models of AD suggest that risk ascertainment and therapeutic interventions in AD as a whole will likely demand complex solutions.

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