We report the cloning, sequence analysis, tissue distribution, functional expression, and chromosomal localization of the human pancreatic sodium bicarbonate cotransport protein (pancreatic NBC (pNBC)). The transporter was identified by searching the human expressed sequence tag data base. An I.M.A.G.E. clone W39298 was identified, and a polymerase chain reaction probe was generated to screen a human pancreas cDNA library. pNBC encodes a 1079-residue polypeptide that differs at the N terminus from the recently cloned human sodium bicarbonate cotransporter isolated from kidney (kNBC) (Burnham, C. E., Amlal, H., Wang, Z., Shull, G. E., and Soleimani, M. (1997) J. Biol. Chem. 272, 19111-19114). Northern blot analysis using a probe specific for the N terminus of pNBC revealed an ϳ7.7-kilobase transcript expressed predominantly in pancreas, with less expression in kidney, brain, liver, prostate, colon, stomach, thyroid, and spinal chord. In contrast, a probe to the unique 5 region of kNBC detected an ϳ7.6-kilobase transcript only in the kidney. In situ hybridization studies in pancreas revealed expression in the acini and ductal cells. The gene was mapped to chromosome 4q21 using fluorescent in situ hybridization. Expression of pNBC in Xenopus laevis oocytes induced sodium bicarbonate cotransport. These data demonstrate that pNBC encodes the sodium bicarbonate cotransporter in the mammalian pancreas. pNBC is also expressed at a lower level in several other organs, whereas kNBC is expressed uniquely in kidney.

Abstract-Recent studies of platelet endothelial cell adhesion molecule-1 (PECAM-1 [CD31])-deficient mice have revealed that this molecule plays an important role in controlling the activation and survival of cells on which it is expressed. In this review, we focus on the complex cytoplasmic domain of PECAM-1 and describe what is presently known about its structure, posttranslational modifications, and binding partners. In addition, we summarize findings that implicate PECAM-1 as an inhibitor of cellular activation via protein tyrosine kinase-dependent signaling pathways, an activator of integrins, and a suppressor of cell death via pathways that depend on damage to the mitochondria. The challenge of future research will be to bridge our understanding of the functional and biochemical properties

Platelet endothelial cell adhesion molecule-1 (PECAM-1, CD31) is a cell adhesion and signaling receptor that is expressed on hematopoietic and endothelial cells. PECAM-1 is vital to the regulation of inflammatory responses, as it has been shown to serve a variety of pro-inflammatory and anti-inflammatory functions. Pro-inflammatory functions of PECAM-1 include the facilitation of leukocyte transendothelial migration and the transduction of mechanical signals in endothelial cells emanating from fluid shear stress. Anti-inflammatory functions include the dampening of leukocyte activation, suppression of pro-inflammatory cytokine production, and the maintenance of vascular barrier integrity. Although PECAM-1 has been well-characterized and studied, the mechanisms through which PECAM-1 regulates these seemingly opposing functions, and how they influence each other, are still not completely understood. The purpose of this review, therefore, is to provide an overview of the pro-and anti-inflammatory functions of PECAM-1 with special attention paid to mechanistic insights that have thus far been revealed in the literature in hopes of gaining a clearer picture of how these opposing functions might be integrated in a temporal and spatial manner on the whole organism level. A better understanding of how inflammatory responses are regulated should enable the development of new therapeutics that can be used in the treatment of acute and chronic inflammatory disorders.

Protein tyrosine phosphorylation controls many aspects of signaling in multicellular organisms. One of the major consequences of tyrosine phosphorylation is the creation of binding sites for proteins containing Src homology 2 (SH2) domains. To profile the global tyrosine phosphorylation state of the cell, we have developed proteomic binding assays encompassing nearly the full complement of human SH2 domains. Here we provide a global view of SH2 domain binding to cellular proteins based on large-scale far-western analyses. We also use reverse-phase protein arrays to generate comprehensive, quantitative SH2 binding profiles for phosphopeptides, recombinant proteins, and entire proteomes. As an example, we profiled the adhesion-dependent SH2 binding interactions in fibroblasts and identified specific focal adhesion complex proteins whose tyrosine phosphorylation and binding to SH2 domains are modulated by adhesion. These results demonstrate that high-throughput comprehensive SH2 profiling provides valuable mechanistic insights into tyrosine kinase signaling pathways.

Previous functional studies have demonstrated that muscle intracellular pH regulation is mediated by sodium-coupled bicarbonate transport, Na ؉ /H ؉ exchange, and Cl ؊ /bicarbonate exchange. We report the cloning, sequence analysis, tissue distribution, genomic organization, and functional analysis of a new member of the sodium bicarbonate cotransporter (NBC) family, NBC3, from human skeletal muscle. mNBC3 encodes a 1214-residue polypeptide with 12 putative membrane-spanning domains. The ϳ 7.8-kilobase transcript is expressed uniquely in skeletal muscle and heart. The NBC3 gene (SLC4A7) spans ϳ80 kb and is composed of 25 coding exons and 24 introns that are flanked by typical splice donor and acceptor sequences. Expression of mNBC3 cRNA in Xenopus laevis oocytes demonstrated that the protein encodes a novel stilbene-insensitive 5-(N-ethyl-N-isopropyl)-amiloride-inhibitable sodium bicarbonate cotransporter.Intracellular pH regulatory mechanisms are critically important for the maintenance of many cellular processes in skeletal muscle, smooth muscle, and myocardial cells (1-8). In muscle cells, contractile processes, metabolic reactions, and membrane transport processes are influenced by pH. Importantly, during periods of increased energy demands and ischemia, muscle cells produce large amounts of lactic acid (3). In these circumstances, intracellular pH (pH i ) 1 regulatory processes prevent the acidification of the sarcoplasm due to lactic acid accumulation.Several different transport mechanisms have been described in muscle cells, which maintain a relatively constant intracellular pH during changes in metabolic proton production or an elevation in ambient CO 2 . The relative contribution of each process varies with cell type, the metabolic requirements of the cell, and local environmental conditions. Intracellular pH regulatory processes that have been characterized functionally in skeletal, smooth muscle, and cardiac cells include: Na ϩ /H ϩ exchange (1-3, 9

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Platelet endothelial cell adhesion molecule-1 (PECAM-1) is a cell surface glycoprotein receptor expressed on a range of blood cells, including platelets, and on vascular endothelial cells. PECAM-1 possesses adhesive and signaling properties, the latter being mediated by immunoreceptor tyrosine-based inhibitory motifs present on the cytoplasmic tail of the protein. Recent studies in vitro have demonstrated that PECAM-1 signaling inhibits the aggregation of platelets. In the present study we have used PECAM-1-deficient mice and radiation chimeras to investigate the function of this receptor in the regulation of thrombus formation. Using intravital microscopy and laserinduced injury to cremaster muscle arterioles, we show that thrombi formed in PECAM-1-deficient mice were larger, formed more rapidly than in control mice, and were more stable. Larger thrombi were also formed in control mice that received transplants of PECAM-1-deficient bone marrow, in comparison to mice that received control transplants. A ferric chloride model of thrombosis was used to investigate thrombus formation in carotid arteries. In PECAM-1-deficient mice the time to 75% vessel occlusion was significantly shorter than in control mice. These data provide evidence for the involvement of platelet PECAM-1 in the negative regulation of thrombus formation. IntroductionPlatelet endothelial cell adhesion molecule-1 (PECAM-1, CD31) is a 130-kDa membrane glycoprotein that is expressed on a range of blood cells including platelets, monocytes, neutrophils, B lymphocytes, some T lymphocyte subsets, and also on vascular endothelial cells. [1][2][3][4] This member of the immunoglobulin superfamily has been reported to be associated with a wide range of functions, depending on the cell of interest. These include transendothelial migration of leukocytes, 5-7 integrin regulation, [8][9][10][11][12][13][14][15][16] modulation of T-and Blymphocyte antigen receptor signaling, 17,18 B-lymphocyte development, 19 vasculogenesis, 20 apoptosis, 21,22 and protection against endotoxic shock. 23 Several lines of investigation have recently determined that PECAM-1 is involved in the negative regulation of platelet function in vitro. The activation of PECAM-1 prior to the stimulation of platelets results in the inhibition of platelet aggregation and the inhibition of activatory signaling mechanisms. 24,25 Of particular note, therefore, are the observations that mouse platelets deficient in PECAM-1 are hyperresponsive to stimulation with collagen and demonstrate enhanced aggregation, secretion, and adhesion to this agonist. 26 Platelets from PECAM-1-deficient mice have also been shown to form larger thrombi in vitro under physiologic flow conditions. 25 PECAM-1 participates in homophilic ligand-binding interactions [27][28][29] ; indeed, such interactions between PECAM-1 molecules on the same cell and between cells are believed to underlie most of its identified functions. Additional potential ligand-binding interactions have been reported, such as with integrin ␣ v ␤ 3 ...

Platelet responses to collagen are mediated by the combined actions of the integrin ␣ 2 ␤ 1 , which serves as a major collagenbinding receptor, and the GPVI/FcR␥-chain complex, which transmits collagen-specific activation signals into the cell interior through the action of an immunoreceptor tyrosine-based activation motif within the cytoplasmic domain of the FcR␥-chain. Despite much progress in identifying components of the signaling pathway responsible for collagen-induced platelet activation, virtually nothing is known about the regulatory elements that modulate this important hemostatic event. PECAM-1, a recently recognized member of the inhibitory receptor family, contains a functional immunoreceptor tyrosine-based inhibitory motif within its cytoplasmic domain that, when tyrosine phosphorylated, recruits and activates the protein-tyrosine phosphatase, SHP-2. To test the hypothesis that PECAM-1 functions to regulate GPVI/FcR␥-chain-mediated platelet activation, the responses of wild-type versus PECAM-1-deficient murine platelets to GPVI-specific agonists were compared. Four distinct GPVI/FcR␥-chain-dependent responses were found to be significantly exaggerated in platelets derived from PE-CAM-1-deficient mice, including Mg ؉؉ -independent adhesion to immobilized fibrillar collagen, collagen-induced platelet aggregation, platelet aggregation induced by the GPVI-specific agonist collagen-related peptide, and GPVI/FcR␥-chain-induced dense granule secretion. Together, these data provide compelling evidence that PECAM-1 modulates platelet responses to collagen, and they implicate this novel member of the inhibitory receptor family in the regulation of primary hemostasis. IntroductionPlatelet endothelial cell adhesion molecule-1 (PECAM-1, CD31) is a 130-kd member of the immunoglobulin (Ig) superfamily that is expressed on the surface of circulating platelets, endothelial cells, neutrophils, monocytes, and certain T-lymphocyte subsets. The extracellular domain of PECAM-1 is composed of 6 extracellular Ig-like homology units, 1 the amino-terminal 2 of which mediates PECAM-1-PECAM-1 homophilic interactions. 2,3 Antibodies to the extracellular domain have been shown to have profound physiologic and cell biologic effects, including delaying leukocyte transendothelial migration 4-6 and inhibiting angiogenesis. 7 The extracellular domain of PECAM-1 also serves as a portal for entry into endothelial cells of certain strains of Plasmodium falciparuminfected erythrocytes. 8,9 The PECAM-1 cytoplasmic domain also plays a key biologic role because a large number of extracellular stimuli (reviewed in Newman 10 ) have been shown to result in the phosphorylation of 2 key tyrosine residues, located at positions 663 and 686 of the cytoplasmic domain. 11 The sequence surrounding each of these 2 tyrosine residues conforms to an immunoreceptor tyrosine-based inhibitory motif (ITIM) that, when phosphorylated, provides a major docking site for the src homology 2 (SH2) domaincontaining protein tyrosine phosphatase (PTP), SHP-2. 11-14 ...