The adenosine 2b receptor is recruited to the plasma membrane and associates with E3KARP and Ezrin upon agonist stimulation

We have previously shown that adenosine is formed in the intestinal lumen during active inflammation from neutrophil-derived 5'-AMP. Acting through the adenosine A2b receptor (A2bR), the luminally derived adenosine induces vectorial chloride secretion and a polarized secretion of interleukin-6 to the intestinal lumen. Although some G protein-coupled receptors interact with anchoring or signaling molecules, not much is known in this critical area for the A2bR. We used the model intestinal epithelial cell line, T84, and Caco2-BBE cells stably transfected with GFP-A2b receptor to study the intestinal A2bR. The A2bR is present in both the apical and basolateral membranes of intestinal epithelia. Apical or basolateral stimulation of the A2bR induces recruitment of the receptor to the plasma membrane and caveolar fractions. The A2bR co-immunoprecipitates with E3KARP and ezrin upon agonist stimulation. Ezrin interacts with E3KARP and PKA and the interaction between ezrin and E3KARP is enhanced by agonist stimulation. Our data suggest that the A2bR is recruited to the plasma membrane upon apical or basolateral agonist stimulation and interacts with E3KARP and ezrin. We speculate that such an interaction may not only anchor the A2bR to the plasma membrane but may also function to stabilize the receptor in a signaling complex in the plasma membrane.     

Adenylyl cyclase 3 mediates prostaglandin E(2)-induced growth inhibition in arterial smooth muscle cells.

Arterial smooth muscle cell (SMC) proliferation contributes to a number of vascular pathologies. Prostaglandin E(2) (PGE(2)), produced by the endothelium and by SMCs themselves, acts as a potent SMC growth inhibitor. The growth-inhibitory effects of PGE(2) are mediated through activation of G-protein-coupled membrane receptors, activation of adenylyl cyclases (ACs), formation of cAMP, and subsequent inhibition of mitogenic signal transduction pathways in SMCs. Of the 10 different mammalian AC isoforms known today, seven isoforms (AC2-7 and AC9) are expressed in SMCs from various species. We show that, despite the presence of several different AC isoforms, the principal AC isoform activated by PGE(2) in human arterial SMCs is a calmodulin kinase II-inhibited AC with characteristics similar to those of AC3. AC3 is expressed in isolated human arterial SMCs and in intact aorta. We further show that arterial SMCs isolated from AC3-deficient mice are resistant to PGE(2)-induced growth inhibition. In summary, AC3 is the principal AC isoform activated by PGE(2) in arterial SMCs, and AC3 mediates the growth-inhibitory effects of PGE(2). Because AC3 activity is inhibited by intracellular calcium through calmodulin kinase II, AC3 may serve as an important integrator of growth-inhibitory signals that stimulate cAMP formation and growth factors that increase intracellular calcium.     

Activation of mitogen-activated protein kinase by the A(2A)-adenosine receptor via a rap1-dependent and via a p21(ras)-dependent pathway.

The A(2A)-adenosine receptor, a prototypical G(s)-coupled receptor, activates mitogen-activated protein (MAP) kinase in a manner independent of cAMP in primary human endothelial cells. In order to delineate signaling pathways that link the receptor to the regulation of MAP kinase, the human A(2A) receptor was heterologously expressed in Chinese hamster ovary (CHO) and HEK293 cells. In both cell lines, A(2A) agonist-mediated cAMP accumulation was accompanied by activation of the small G protein rap1. However, rap1 mediates A(2A) receptor-dependent activation of MAP kinase only in CHO cells, the signaling cascade being composed of G(s), adenylyl cyclase, rap1, and the p68 isoform of B-raf. This isoform was absent in HEK293 cells. Contrary to CHO cells, in HEK293 cells activation of MAP kinase by A(2A) agonists was not mimicked by 8-bromo-cAMP, was independent of Galpha(s), and was associated with activation of p21(ras). Accordingly, overexpression of the inactive S17N mutant of p21(ras) and of a dominant negative version of mSos (the exchange factor of p21(ras)) blocked MAP kinase stimulation by the A(2A) receptor in HEK 293 but not in CHO cells. In spite of the close homology between p21(ras) and rap1, the S17N mutant of rap1 was not dominant negative because (i) overexpression of rap1(S17N) failed to inhibit A(2A) receptor-dependent MAP kinase activation, (ii) rap1(S17N) was recovered in the active form with a GST fusion protein comprising the rap1-binding domain of ralGDS after A(2A) receptor activation, and (iii) A(2A) agonists promoted the association of rap1(S17N) with the 68-kDa isoform of B-raf in CHO cells. We conclude that the A(2A) receptor has the capacity two activate MAP kinase via at least two signaling pathways, which depend on two distinct small G proteins, namely p21(ras) and rap1. Our observations also show that the S17N version of rap1 cannot be assumed a priori to act as a dominant negative interfering mutant.     

Chemokine receptor CCR6 transduces signals that activate p130Cas and alter cAMP-stimulated ion transport in human intestinal epithelial cells

Human colon epithelial cells express the G protein-coupled receptor CCR6, the sole receptor for the chemokine CCL20 (also termed MIP-3). CCL20 produced by intestinal epithelial cells is upregulated in response to proinflammatory stimuli and microbial infection, and it chemoattracts leukocytes, including CCR6-expressing immature myeloid dendritic cells, into sites of inflammation. The aim of this study was to determine whether CCR6 expressed by intestinal epithelial cells acts as a functional receptor for CCL20 and whether stimulation with CCL20 alters intestinal epithelial cell functions. The human colon epithelial cell lines T84, Caco-2, HT-29, and HCA-7 were used to model colonic epithelium. Polarized intestinal epithelial cells constitutively expressed CCR6, predominantly on the apical side. Consistent with this, apical stimulation of polarized intestinal epithelial cells resulted in tyrosine phosphorylation of the p130 Crk-associated substrate (Cas), an adaptor/scaffolding protein that localizes in focal adhesions and has a role in regulating cytoskeletal elements important for cell attachment and migration. In addition, CCL20 stimulation inhibited agonist-stimulated production of the second messenger cAMP and cAMP-mediated chloride secretory responses by intestinal epithelial cells. Inhibition was abrogated by pertussis toxin, consistent with signaling through G_i proteins that negatively regulate adenylyl cyclases and cAMP production. These data indicate that signaling events, occurring via the activation of the apically expressed chemokine receptor CCR6 on polarized intestinal epithelial cells, alter specialized intestinal epithelial cell functions, including electrogenic ion secretion and possibly epithelial cell adhesion and migration. CCL20; macrophage inflammatory protein-3; forskolin; G protein-coupled receptors; tyrosine phosphorylation     

Angiotensin II enhances adenylyl cyclase signaling via Ca2+/calmodulin. Gq-Gs cross-talk regulates collagen production in cardiac fibroblasts

Cardiac fibroblasts regulate formation of extracellular matrix in the heart, playing key roles in cardiac remodeling and hypertrophy. In this study, we sought to characterize cross-talk between Gq and Gs signaling pathways and its impact on modulating collagen synthesis by cardiac fibroblasts. Angiotensin II (ANG II) activates cell proliferation and collagen synthesis but also potentiates cyclic AMP (cAMP) production stimulated by beta-adrenergic receptors (beta-AR). The potentiation of beta-AR-stimulated cAMP production by ANG II is reduced by phospholipase C inhibition and enhanced by overexpression of Gq. Ionomycin and thapsigargin increased intracellular Ca2+ levels and potentiated isoproterenol- and forskolin-stimulated cAMP production, whereas chelation of Ca2+ with 1,2-bis(2-aminophenoxy)ethane-N,N,N', N'-tetraacetic acid/AM inhibited such potentiation. Inhibitors of tyrosine kinases, protein kinase C, or Gbetagamma did not alter this cross-talk. Immunoblot analyses showed prominent expression of adenylyl cyclase 3 (AC3), a Ca2+-activated isoform, along with AC2, AC4, AC5, AC6, and AC7. Of those isoforms, only AC3 and AC5/6 proteins were detected in caveolin-rich fractions. Overexpression of AC6 increased betaAR-stimulated cAMP accumulation but did not alter the size of the ANG II potentiation, suggesting that the cross-talk is AC isoform-specific. Isoproterenol-mediated inhibition of serum-stimulated collagen synthesis increased from 31 to 48% in the presence of ANG II, indicating that betaAR-regulated collagen synthesis increased in the presence of ANG II. These data indicate that ANG II potentiates cAMP formation via Ca2+-dependent activation of AC activity, which in turn attenuates collagen synthesis and demonstrates one functional consequence of cross-talk between Gq and Gs signaling pathways in cardiac fibroblasts.     

Vector-based RNAi,a novel tool for isoform-specific knock-down of VEGF and anti-angiogenesis gene therapy of cancer

Vascular endothelial growth factor (VEGF) carries out multifaceted functions in tumor development, and it exists as at least five isoforms with distinct biologic activities and clinical implications. Several strategies have been developed to block VEGF for cancer therapy; however, the approach to target-specific VEGF isoform(s) has not been explored to date. In the present study, we show that DNA vector-based RNA interference (RNAi), in which RNAi sequences targeting murine VEGF isoforms are inserted downstream of an RNA polymerase III promoter, has potential applications in isoform-specific "knock-down" of VEGF. Large molecular weight VEGF isoforms were specifically reduced in vitro in the presence of isoform-specific RNAi constructs. Additionally, H1 promoter may be superior to U6 promoter when used for vector-based RNAi of VEGF isoforms. This strategy provides a novel tool to study the function of various VEGF isoforms and may contribute to VEGF isoform-specific treatment in cancer.     

Distribution of CTP:phosphocholine cytidylyltransferase (CCT) isoforms. Identification of a new CCTbeta splice variant.

CTP:phosphocholine cytidylyltransferase is a major regulator of phosphatidylcholine biosynthesis. A single isoform, CCTalpha, has been studied extensively and a second isoform, CCTbeta, was recently identified. We identify and characterize a third cDNA, CCTbeta2, that differs from CCTbeta1 at the carboxyl-terminal end and is predicted to arise as a splice variant of the CCTbeta gene. Like CCTalpha, CCTbeta2 is heavily phosphorylated in vivo, in contrast to CCTbeta1. CCTbeta1 and CCTbeta2 mRNAs were differentially expressed by the human tissues examined, whereas CCTalpha was more uniformly represented. Using isoform-specific antibodies, both CCTbeta1 and CCTbeta2 localized to the endoplasmic reticulum of cells, in contrast to CCTalpha which resided in the nucleus in addition to associating with the endoplasmic reticulum. CCTbeta2 protein has enzymatic activity in vitro and was able to complement the temperature-sensitive cytidylyltransferase defect in CHO58 cells, just as CCTalpha and CCTbeta1 supporting proliferation at the nonpermissive conditions. Overexpression experiments did not reveal discrete physiological functions for the three isoforms that catalyze the same biochemical reaction; however, the differential cellular localization and tissue-specific distribution suggest that CCTbeta1 and CCTbeta2 may play a role that is distinct from ubiquitously expressed CCTalpha.     

Distinct targeting and recycling properties of two isoforms of the iron transporter DMT1 (NRAMP2, Slc11A2)

The metal transporter DMT1 (Slc11a2) plays a vital role in iron metabolism. Alternative splicing of the 3' exon generates two DMT1 isoforms with different C-terminal protein sequences and a 3' untranslated region harboring (isoform I, +IRE) or not (isoform II, -IRE), an iron-responsive element. Isoform I is expressed at the plasma membrane of certain epithelial cells including the duodenum brush border, where it is essential for the absorption of nutritional iron. Isoform II is expressed in many cells and is essential for the acquisiton of transferrin iron from acidified endosomes. The targeting and trafficking properties of DMT1 isoforms I and II were studied in transfected LLC-PK(1) kidney cells, with respect to isoform-specific differences in function, subcellular localization, endocytosis kinetics, and fate upon internalization. Isoform I showed higher surface expression and was internalized from the plasma membrane with slower kinetics than that of isoform II. As opposed to isoform II, which is efficiently sorted to recycling endosomes upon internalization, isoform I was not efficiently recycled and was targeted to lysosomes. Thus, alternative splicing of DMT1 critically regulates the subcellular localization and site of Fe(2+) transport.     

SDF-1/CXCL12 regulates cAMP production and ion transport in intestinal epithelial cells via CXCR4

Human colonic epithelial cells express CXCR4, the sole cognate receptor for the chemokine stromal cell-derived factor (SDF)-1/CXC chemokine ligand (CXCL) 12. The aim of this study was to define the mechanism and functional consequences of signaling intestinal epithelial cells through the CXCR4 chemokine receptor. CXCR4, but not SDF-1/CXCL12, was constitutively expressed by T84, HT-29, HT-29/-18C1, and Caco-2 human colon epithelial cell lines. Studies using T84 cells showed that CXCR4 was G protein-coupled in intestinal epithelial cells. Moreover, stimulation of T84 cells with SDF-1/CXCL12 inhibited cAMP production in response to the adenylyl cyclase activator forskolin, and this inhibition was abrogated by either anti-CXCR4 antibody or receptor desensitization. Studies with pertussis toxin suggested that SDF-1/CXCL12 activated negative regulation of cAMP production through G(i)alpha subunits coupled to CXCR4. Consistent with the inhibition of forskolin-stimulated cAMP production, SDF-1/CXCL12 also inhibited forskolin-induced ion transport in voltage-clamped polarized T84 cells. Taken together, these data indicate that epithelial CXCR4 can transduce functional signals in human intestinal epithelial cells that modulate important cAMP-mediated cellular functions.     

Novel mechanism of cyclic AMP mediated extracellular signal regulated kinase activation in an intestinal cell line

The extracellular signal regulated kinase (ERK1/2) signaling cascade has been implicated as both a pro-apoptotic and anti-apoptotic pathway depending on cell type and context. In the T84 intestinal epithelial cell line, cAMP activates ERK1/2 resulting in the inhibition of apoptosis. Cyclic-AMP signaling relies on the binding and activation of a cAMP binding protein. In most cell types, the majority of this signaling occurs through an isoform of protein kinase A (PKAI or PKAII). Despite evidence to the contrary, we hypothesized that ERK1/2 activation is through a PKA isoform. Pharmacological activators and inhibitors of PKA as well as siRNA were used to further interrogate this potential signaling pathway. Our results demonstrate that at doses sufficient to increase PKA activity, PKAII specific cAMP analogs activate ERK1/2 while PKAI analogs do not. Pharmacological inhibition of the PKAII regulatory subunit and catalytic subunit as well as siRNA knockdown of the catalytic subunit blocks ERK1/2 activation. We conclude that in the T84 cell line, cAMP binding to the PKAII regulatory subunit leads to the subsequent phosphorylation of ERK1/2 and provides insight into the mechanism of cAMP mediated survival signaling in the intestinal epithelium. These results directly implicate PKAII as a mediator of cell survival in T84 cells and provide evidence for an additional means by which cAMP can influence intestinal cell turnover.     

Regulation of ERK1/2 by the C. elegans muscarinic acetylcholine receptor GAR-3 in Chinese hamster ovary cells

Three G-protein-linked acetylcholine receptors (GARs) exist in the nematode C. elegans. GAR-3 is pharmacologically most similar to mammalian muscarinic acetylcholine receptors (mAChRs). We observed that carbachol stimulated ERK1/2 activation in Chinese hamster ovary (CHO) cells stably expressing GAR-3b, the predominant alternatively spliced isoform of GAR-3. This effect was substantially reduced by the phospholipase C (PLC) inhibitor U73122 and the protein kinase C (PKC) inhibitor GF109203X, implying that PLC and PKC are involved in this process. On the other hand, GAR-3b-mediated ERK1/2 activation was inhibited by treatment with forskolin, an adenylate cyclase (AC) activator. This inhibitory effect was blocked by H89, an inhibitor of cAMP-dependent protein kinase A (PKA). These results suggest that GAR-3b-mediated ERK1/2 activation is negatively regulated by cAMP through PKA. Together our data show that GAR-3b mediates ERK1/2 activation in CHO cells and that GAR-3b can couple to both stimulatory and inhibitory pathways to modulate ERK1/2.     

Agonist-induced polarized trafficking and surface expression of the adenosine 2b receptor in intestinal epithelial cells: role of SNARE proteins

Adenosine, acting through the A2b receptor, induces vectorial chloride and IL-6 secretion in intestinal epithelia and may play an important role in intestinal inflammation. We have previously shown that apical or basolateral adenosine receptor stimulation results in the recruitment of the A2b receptor to the plasma membrane. In this study, we examined domain specificity of recruitment and the role of soluble N-ethylmaleimide (NEM) attachment receptor (SNARE) proteins in the agonist-mediated recruitment of the A2b receptor to the membrane. The colonic epithelial cell line T84 was used because it only expresses the A2b-subtype adenosine receptor. Cell fractionation, biotinylation, and electron microscopic studies showed that the A2b receptor is intracellular at rest and that apical or basolateral adenosine stimulation resulted in the recruitment of the receptor to the apical membrane. Upon agonist stimulation, the A2b receptor is enriched in the vesicle fraction containing vesicle-associated membrane protein (VAMP)-2. Furthermore, in cells stimulated with apical or basolateral adenosine, we demonstrate a complex consisting of VAMP-2, soluble NEM-sensitive factor attachment protein (SNAP)-23, and A2b receptor that is coimmunoprecipitated in cells stimulated with adenosine within 5 min and is no longer detected within 15 min. Inhibition of trafficking with NEM or nocodazole inhibits cAMP synthesis induced by apical or basolateral adenosine by 98 and 90%, respectively. cAMP synthesis induced by foskolin was not affected, suggesting that generalized signaling is not affected under these conditions. Collectively, our data suggest that 1) the A2b receptor is intracellular at rest; 2) apical or basolateral agonist stimulation induces recruitment of the A2b receptor to the apical membrane; 3) the SNARE proteins, VAMP-2 and SNAP-23, participate in the recruitment of the A2b receptor; and 4) the SNARE-mediated recruitment of the A2b receptor may be required for its signaling.     

Lamina-associated polypeptide 2 (LAP2) expression in fish and amphibians

Somatic and germinal cells of 15 fish and 33 amphibian species were examined by SDS-PAGE followed by immunoblotting to determine the expression of LAP2 (lamina-associated polypeptide 2). LAP2 expression in frogs, salamanders and fish does not vary with the mode of reproduction. In fish and frog cells, a rim-like LAP2 positive region was detected around the nucleus by indirect immunofluorescence microscopy. The cell distribution and expression patterns of LAP2 in fish, frogs and salamanders are comparable with those found in Xenopus and zebrafish. The mammalian somatic cell pattern, which may also occur in gymnophione amphibians, includes LAP2alpha, beta and gamma as major isoforms, whereas LAP2alpha does not occur in cells of fish, frogs and salamanders. In fish, LAP2gamma is the major isoform of somatic cells, suggesting that LAP2gamma may be ancestral. However, in the rainbow trout, as in frogs and salamanders, LAP2beta was the major somatic isoform. Fish and frog sperm only express low molecular weight polypeptides. In contrast, fish and frog oocytes express an oocyte-specific LAP2 isoform of high molecular weight. In the toad Bufo marinus this isoform becomes upregulated in pre-vitellogenic oocytes of 150-200 microm in diameter. The absence of LAP2alpha and the differential expression of LAP2 isoforms in somatic and germ cells, as found in fish and frogs, may be ancestral vertebrate characters. In spite of differences in developmental time, the LAP2 isoforms of somatic cells are upregulated during gastrulation, suggesting that LAP2 may be implicated in the early development of fish and frog.     

VPAC2在CHO细胞的表达及鉴定

PAC2是垂体腺苷酸环化酶激活多肽(Pituitary adenylate cyclase activating polypeptide,PACAP)和血管活性肠肽(vasoactive intestinal peptide,VIP)的共同受体,介导多种重要生物学功能。为获得稳定特异表达VPAC2的中国仓鼠卵巢(Chinesehamsterovary,CHO)细胞,将pcDNA-VPAC2表达载体转染CHO细胞,G418筛选转染阳性克隆,PACAP38标准品诱导阳性克隆细胞的胞内cAMP生成,筛选出对PACAP38最为敏感的阳性单克隆细胞株(VPAC2-CHO),运用RT-PCR、Westernblot和免疫荧光法检测VPAC2受体表达情况,利用VPAC2受体特异激动剂通过竞争性结合试验和促进胞内第二信使cAMP生成的活性检测实验证实,VPAC2-CHO特异表达有功能的VPAC2。Scatchard作图分析显示VPAC2-CHO的VPAC2受体密度为(1.1±0.2)pmol/mg膜蛋白,PACAP38与VPAC2的解离常数Kd值为(0.55±0.10)nmol/L。特异表达VPAC2受体细胞系的构建为深入研究该受体理化性质、生物学功能以及筛选、开发VPAC2受体新型特异激动剂和拮抗剂等研究奠定了基础。     

Mouse proton pump ATPase C subunit isoforms (C2-a and C2-b) specifically expressed in kidney and lung

The vacuolar-type H+-ATPases (V-ATPases) are multimeric proton pumps involved in a wide variety of physiological processes. We have identified two alternative splicing variants of C2 subunit isoforms: C2-a, a lung-specific isoform containing a 46-amino acid insertion, and C2-b, a kidney-specific isoform without the insert. Immunohistochemistry with isoform-specific antibodies revealed that V-ATPase with C2-a is localized specifically in lamellar bodies of type II alveolar cells, whereas the C2-b isoform is found in the plasma membranes of renal alpha and beta intercalated cells. Immunoprecipitation combined with immunohistological analysis revealed that C2-b together with other kidney-specific isoforms was selectively assembled to form a unique proton pump in intercalated cells. Furthermore, a chimeric yeast V-ATPase with mouse the C2-a or C2-b isoform showed a lower Km(ATP) and lower proton transport activity than that with C1 or Vma5p (yeast C subunit). These results suggest that V-ATPases with the C2-a and C2-b isoform are involved in luminal acidification of lamellar bodies and regulation of the renal acid-base balance, respectively.     

Cholangiocyte primary cilia are chemosensory organelles that detect biliary nucleotides via P2Y12 purinergic receptors

Cholangiocytes, the epithelial cells lining intrahepatic bile ducts, contain primary cilia, which are mechano- and osmosensory organelles detecting changes in bile flow and osmolality and transducing them into intracellular signals. Here, we asked whether cholangiocyte cilia are chemosensory organelles by testing the expression of P2Y purinergic receptors and components of the cAMP signaling cascade in cilia and their involvement in nucleotide-induced cAMP signaling in the cells. We found that P2Y(12) purinergic receptor, adenylyl cyclases (i.e., AC4, AC6, and AC8), and protein kinase A (i.e., PKA RI-beta and PKA RII-alpha regulatory subunits), exchange protein directly activated by cAMP (EPAC) isoform 2, and A-kinase anchoring proteins (i.e., AKAP150) are expressed in cholangiocyte cilia. ADP, an endogenous agonist of P2Y(12) receptors, perfused through the lumen of isolated rat intrahepatic bile ducts or applied to the ciliated apical surface of normal rat cholangiocytes (NRCs) in culture induced a 1.9- and 1.5-fold decrease of forskolin-induced cAMP levels, respectively. In NRCs, the forskolin-induced cAMP increase was also lowered by 1.3-fold in response to ATP-gammaS, a nonhydrolyzed analog of ATP but was not affected by UTP. The ADP-induced changes in cAMP levels in cholangiocytes were abolished by chloral hydrate (a reagent that removes cilia) and by P2Y(12) siRNAs, suggesting that cilia and ciliary P2Y(12) are involved in nucleotide-induced cAMP signaling. In conclusion, cholangiocyte cilia are chemosensory organelles that detect biliary nucleotides through ciliary P2Y(12) receptors and transduce corresponding signals into a cAMP response.     

Coupling of thromboxane A2 receptor isoforms to Galpha13: effects on ligand binding and signalling.

Previous subtyping of thromboxane A2 (TXA2) receptors in platelets and vascular smooth muscle cells was based on pharmacological criteria. Two distinct carboxy-terminal splice variants for TXA2 receptors exist and they couple to several different G protein alpha subunits including Galpha13, but it has not been established whether either or both isoforms interact with and signal through it. We sought to determine: (1) which TXA2 receptor isoforms exist in vascular smooth muscle, (2) if Galpha13 is present in vascular smooth muscle and (3) if Galpha13 interacts with either or both of the two TXA2 receptor isoforms as determined by changes in ligand binding properties and generation of intracellular signals. Both TXA2 receptor isoforms and Galpha13 were found in vascular smooth muscle cells. Both the alpha and beta isoforms of the TXA2 receptors were transiently transfected with or without Galpha13 into COS-7 (radioligand binding assays) or CHO cells (agonist induced Na+/H+ exchange). Co-expression of each receptor isoform with Galpha13 significantly (P<0.05) increased the affinity of each receptor for the two agonists, I-BOP and ONO11113, and decreased the affinity of the receptor for the antagonists, SQ29,548 and L657,925. I-BOP stimulated Na+/H+ exchange in vascular smooth muscle cells. Co-expression of Galpha13 with each TXA2 receptor isoform in CHO cells resulted in a significant (P<0.04) agonist induced increase in Na+/H+ exchange compared to cells not transfected with Galpha13. The results support the possibility that the previous classification of TXA2 receptor subtypes based on pharmacological criteria reflect unique interactions with specific G protein alpha subunits.