Multiple P2Y receptor subtypes in the apical membranes of polarized epithelial cells

McAlroy, H. L.; Ahmed, SG; Day, Sharlene M.; Baines, Deborah L.; Wong, Hau Yan; Yip, Chung-Yin; Ko, Wing‐Hung; Wilson, Samantha M.; Collett, Andrew

doi:10.1038/sj.bjp.0703743

Cited by 31 publications

(24 citation statements)

References 30 publications

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“…It is not widely appreciated outside the specialist kinase field that AMPK has a substantial and constitutive baseline activity in most tissues that is further stimulated by increments in the intracellular AMP/ATP ratio or pharmacological activation by drugs such as phenformin. Additionally, independent regulation by the calmodulin-dependent kinase (CAMKK) has been shown previously (27,28). It may be of relevance to our findings in oocytes that PKA was shown to inhibit AMPK activity by attenuating phosphorylation through upstream calcium/calmodulindependent protein kinase (29).…”

Section: Discussionsupporting

confidence: 54%

Mechanistic Insight into Control of CFTR by AMPK

Kongsuphol

Cassidy

Hieke

et al. 2009

Journal of Biological Chemistry

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Section: Discussionsupporting

confidence: 54%

Mechanistic Insight into Control of CFTR by AMPK

Kongsuphol

Cassidy

Hieke

et al. 2009

Journal of Biological Chemistry

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“…Moreover, P2Y 2 receptor activation increases mucous secretion and ciliary beat frequency (19). Thus, activation of the P2Y 2 receptor facilitates mucociliary clearance and is an important therapeutic target for treatment of cystic fibrosis and other diseases of epithelia (20).We have recently demonstrated by confocal microscopy that the P2Y 2 receptor is targeted to the apical membrane in epithelial cells of multiple lineages (21), and this localization is consistent with pharmacological studies of the receptor in epithelial cell lines (22,23). Proteins are targeted to a particular membrane surface because they contain a signal within their primary sequence that is recognized by the cell.…”

supporting

confidence: 60%

“…We have recently demonstrated by confocal microscopy that the P2Y 2 receptor is targeted to the apical membrane in epithelial cells of multiple lineages (21), and this localization is consistent with pharmacological studies of the receptor in epithelial cell lines (22,23). Proteins are targeted to a particular membrane surface because they contain a signal within their primary sequence that is recognized by the cell.…”

mentioning

confidence: 71%

The Apical Targeting Signal of the P2Y2 Receptor Is Located in Its First Extracellular Loop

Wolff

Nicholas

2005

Journal of Biological Chemistry

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P2Y 2 and P2Y 4 receptors, which have 52% sequence identity, are both expressed at the apical membrane of Madin-Darby canine kidney cells, but the locations of their apical targeting signals are distinctly different. The targeting signal of the P2Y 2 receptor is located between the N terminus and 7TM, whereas that of the P2Y 4 receptor is present in its C-terminal tail. To identify the apical targeting signal in the P2Y 2 receptor, regions of the P2Y 2 receptor were progressively substituted with the corresponding regions of the P2Y 4 receptor lacking its targeting signal. Characterization of these chimeras and subsequent mutational analysis revealed that four amino acids (Arg 95 , Gly 96 , Asp 97 , and Leu 108 ) in the first extracellular loop play a major role in apical targeting of the P2Y 2 receptor. Mutation of RGD to RGE had no effect on P2Y 2 receptor targeting, indicating that receptor-integrin interactions are not involved in apical targeting. P2Y 2 receptor mutants were localized in a similar manner in Caco-2 colon epithelial cells. This is the first identification of an extracellular protein-based targeting signal in a seven-transmembrane receptor.Extracellular nucleotides play an important role in a broad range of physiological and pathophysiological processes through two classes of receptors, the ligand-gated P2X receptors and the G protein-coupled P2Y receptors (1, 2). To date, pharmacological characterization and molecular cloning have identified seven P2X receptors (P2X 1-7 ) and eight P2Y receptor subtypes (P2Y 1 , P2Y 2 , P2Y 4 , P2Y 6 , P2Y 11 , P2Y 12 , P2Y 13 , and P2Y 14 ). The P2Y receptors can be divided into two main subfamilies based on sequence identity and phylogenetic considerations: the P2Y 1 receptor subfamily, which is comprised of P2Y 1 , P2Y 2 , P2Y 4 , P2Y 6 , and P2Y 11 receptors, and the P2Y 12 receptor subfamily, comprising P2Y 12 , P2Y 13 , and P2Y 14 receptors. The P2Y 1 receptor subfamily is coupled to activation of phospholipase C, generation of inositol phosphates, activation of protein kinase C, and mobilization of intracellular Ca 2ϩ stores. In addition to coupling to activation of phospholipase C, the P2Y 11 receptor is also coupled to G s , thereby activating adenylyl cyclase (3-5). In contrast, the three members of the P2Y 12 receptor subfamily couple to G i/o and therefore inhibit adenylyl cyclase (6 -8).Although P2Y receptors mediate a multitude of cellular responses throughout the body, one of their main functions is to regulate ion transport in polarized epithelial cells. Proteins in polarized epithelial cells can be expressed predominantly at either the apical or basolateral membrane surface, or they can be indiscriminatingly distributed to both membranes. The proper targeting of proteins to their respective membrane surface is critical in host defense, nutrient absorption, ion transport, and signal transduction. However, the mechanism(s) by which epithelial cells target proteins to a particular membrane surface remains a critical question in epithelial cell b...

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“…Certain of the receptors (e.g. P2Y 12 , P2Y 13 ) are known to utilize G i -dependent mechanisms to inhibit cAMP formation (29,30), whereas other P2Y receptors may regulate adenylyl cyclase activity via effects on regulators of adenylyl cyclase, such as [Ca 2ϩ ] or protein kinase C. MDCK-D 1 cells, in common with several other cell types, express several different P2Y receptor subtypes (1,6,12,31,32). Early work on MDCK-D 1 cells, prior to the molecular cloning and precise identification of the different P2Y receptor subtypes, emphasized the ability of added nucleotides to alter membrane potential, ion flux, or short circuit current (7,8).…”

Section: Resultsmentioning

confidence: 99%

P2Y11 Receptors Activate Adenylyl Cyclase and Contribute to Nucleotide-promoted cAMP Formation in MDCK-D1Cells

Torres

Zambon

Insel

2002

Journal of Biological Chemistry

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Cells commonly co-express multiple receptor subtypes that recognize the same physiological agonist, but it is difficult to define which among such receptor subtypes mediates a particular response. This can be a particularly vexing problem if subtype-selective agonists and antagonists are not available. One such example is P2Y receptors, which respond to ATP and other nucleotides, are expressed in a variety of tissues and cell types, and for which few subtype-selective antagonists exist (2-4). Our laboratory has undertaken a series of studies related to signal transduction by P2Y receptors in the renal epithelial cell line, MDCK 1 -D 1 (reviewed in Refs. 5 and 6). P2Y receptors in MDCK-D 1 cells modulate membrane potential and short circuit current, and the receptors regulate phospholipases, intracellular [Ca 2ϩ ], prostaglandin E 2 (PGE 2 ) formation, and cAMP accumulation (1,(7)(8)(9)(10)(11)(12)(13)(14).Cyclooxygenase (COX) inhibitors, such as indomethacin (indo), have proven useful for studying P2Y receptors in MDCK-D 1 cells (1, 12). Agonist-stimulated cAMP accumulation in MDCK-D 1 cells occurs via both indo-sensitive and -insensitive pathways. Response to the P2Y 2 agonist UTP is entirely indo-sensitive, whereas response to ATP is partially sensitive and to 2-methylthio-ATP (MT-ATP) is insensitive (1). These findings suggest that UTP, and ATP in part, stimulate P2Y 2 receptors to cause COX-mediated (perhaps by both COX1 and COX2, see Ref. 15) formation of arachidonic acid metabolites (e.g. PGE 2 ), which activate EP receptors to stimulate cAMP formation, while ATP and MT-ATP can also enhance cAMP formation via an indo-insensitive P2Y receptor pathway (1).The present studies were designed to characterize more fully the nature of the latter pathway. Our working hypothesis, based in part on initial results obtained with MDCK-D 1 cells (12), was that the indo-resistant response might represent a P2Y 1 receptor effect. The current data show results not consistent with this hypothesis but instead suggest a key role for another receptor, the P2Y 11 receptor, in indo-resistant cAMP formation. The findings directly document a role for P2Y 11 receptors in stimulation of adenylyl cyclase activity and in potentially contributing to autocrine-paracrine regulation by nucleotides. EXPERIMENTAL PROCEDURESCell Culture-MDCK-D 1 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% mixed serum (85% horse serum, 15% fetal bovine serum). Cells were used in assays at 60 -80% confluency. GFP-tagged cP2Y 11 receptor-overexpressing MDCK-D 1 cells were cultured from the stable cell line prepared by Zambon et al. (14).Measurement of cAMP Accumulation in Normal and GFP-tagged cP2Y 11 Intact Cells-Prior to the treatment of the cells, the growth medium was removed, and cells were equilibrated for 30 min at 37°C in serum-free 20 mM HEPES-buffered Dulbecco's modified Eagle's medium (DMEH, pH 7.4). Subsequently, cells were incubated in fresh DMEH and various agents as shown in the figures. Incubations with th...

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Multiple P2Y receptor subtypes in the apical membranes of polarized epithelial cells

Cited by 31 publications

References 30 publications

Mechanistic Insight into Control of CFTR by AMPK

Mechanistic Insight into Control of CFTR by AMPK

The Apical Targeting Signal of the P2Y2 Receptor Is Located in Its First Extracellular Loop

P2Y11 Receptors Activate Adenylyl Cyclase and Contribute to Nucleotide-promoted cAMP Formation in MDCK-D1Cells

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