P2Y ₁ receptor signaling is controlled by interaction with the PDZ scaffold NHERF-2

Abstract

P2Y1 purinergic receptors (P2Y1Rs) mediate rises in intracellular Ca 2؉ in response to ATP, but the duration and characteristics of this Ca 2؉ response are known to vary markedly in distinct cell types. We screened the P2Y1R carboxyl terminus against a recently created proteomic array of PDZ (PSD-95͞Drosophila Discs large͞ZO-1 homology) domains and identified a previously unrecognized, specific interaction with the second PDZ domain of the scaffold NHERF-2 (Na ؉ ͞H ؉ exchanger regulatory factor type 2). Furthermore, we found that P2Y1R and NHERF-2 associate in cells, allowing NHERF-2-mediated tethering of P2Y 1R to key downstream effectors such as phospholipase C␤. Finally, we found that coexpression of P2Y1R with NHERF-2 in glial cells prolongs P2Y1R-mediated Ca 2؉ signaling, whereas disruption of the P2Y1R-NHERF-2 interaction by point mutations attenuates the duration of P2Y1R-mediated Ca 2؉ responses. These findings reveal that NHERF-2 is a key regulator of the cellular activity of P2Y 1R and may therefore determine cellspecific differences in P2Y1R-mediated signaling.G protein-coupled receptor ͉ purinergic ͉ ATP ͉ proteomic array A denine-based nucleotides such as ATP and ADP are prominent extracellular signaling molecules that mediate a wide variety of physiological actions in tissues throughout the body. Many of the physiological effects evoked by ATP and ADP are mediated by metabotropic P2Y receptors (P2YRs) (1), which are members of the G protein-coupled receptor (GPCR) superfamily. To date, seven distinct mammalian P2YR subtypes have been cloned: P2Y 1 , P2Y 2 , P2Y 4 , P2Y 6 , P2Y 11 , P2Y 12 , and P2Y 13 . Most P2YRs are coupled to G␣ q proteins and thus to the activation of phospholipase C␤ (PLC␤) and generation of diacylglycerol and inositol-3,4,5-trisphosphate, ultimately leading to the activation of PKC and release of Ca 2ϩ from internal stores (1). Some P2YRs, including P2Y 1 , P2Y 2 , P2Y 12 , and P2Y 13 , are also known to couple to G␣ i and the inhibition of adenylyl cyclase (2-5). The purinergic P2YR type 1 (P2Y 1 R) subtype is abundantly expressed in a number of tissues, including the CNS (6, 7), where it plays a key role in the transmission of astrocytic Ca 2ϩ waves (8), activation of mitogenic responses in astrocytes to brain trauma (9), inhibition of neuronal N-type voltage-activated Ca 2ϩ channels (10), and embryonic brain development (11). P2Y 1 R also plays critical roles in the cardiovascular system, including the regulation of coronary vasodilation (12) and platelet aggregation (13).Signaling by P2Y 1 Rs is known to be heavily dependent on cellular context. For example, stimulation of P2Y 1 Rs in some cell types is known to strongly promote cell proliferation (14), whereas P2Y 1 R stimulation in other cell types is known to induce apoptosis (15). Furthermore, P2Y 1 Rs can exert cellular effects that are quite different from those exerted by other P2YRs expressed in the same cell type and that couple to similar G proteins. For example, in astrocytes, both P2Y 1 Rs and P2Y 2 Rs can cou...

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“…LPA 2 has carboxyl-terminal motifs that allow interaction with a PDZ (PSD-95/DlgA/ZO-1) domain of the Na + /H + exchanger regulatory factor 2 (NHERF2), which functions as a scaffold to link signaling proteins, such as PKA and PKCα, to GPCRs and transporters at the cell surface [21,23,[29][30][31]. NHERF2 links LPA 2 to PLC-β3 and hence affects the activation of downstream targets, such as Erk and cyclooxygenase-2 [23].…”

Postnatal developmental expression of the PDZ scaffolds Na+-H+ exchanger regulatory factors 1 and 2 in the rat cochlea

“…It is clear from the literature that NHERFs play key roles in mediating a number of transport and signaling phenomena by forming macromolecular complexes of transporters, ion channels, receptors, and structural proteins (Hall et al 1998;Fanning and Anderson 1999;Lederer et al 2003;Pushkin et al 2003;Yun 2003;Fam et al 2005;Weinman et al 2005). The rationale for the current study comes from the finding that the cochlea contains a number of proteins known to interact with NHERFs, plus numerous other proteins that have PDZ domains that could potentially interact with NHERFs.…”

“…NHERF-1 and ERM proteins have also been reported in polarized Schwann cell processes where they have been implicated in the formation of the nodes of Ranvier (Gatto et al 2003). NHERF-2 has recently been shown to co-localize with P2Y1 in glial cells, in which NHERF-2 interaction regulates P2Y1-mediated Ca 2+ signaling (Fam et al 2005). Thus, the presence of NHERF-1 and NHERF-2 in the glial cells located in the cochlear nerve and the spiral ganglion (satellite cells) is in keeping with its known expression in other glial cells.…”

“…Extracellular ATP affects cochlear function by influencing the endocochlear potential, micromechanics, and neurotransmission via P2X and P2Ypurinergic receptors (Housley et al 1999;Lee et al 2001;Housley et al 2002). The interaction of NHERF-2 with the PDZ-binding motif containing P2Y1 receptors (Hall et al 1998;Fam et al 2005) and the localization of P2Y1 receptors in sensory, supporting (e.g., Deiters cells and glia), and secretory cells of the cochlea (Teixeira et al 2000), in which we have shown NHERF-2 expression, indicate a potential role for NHERFs in the modulation of purinergic signaling.…”

“…NHERF-1 and NHERF-2 bind PDZ motifs in the C-termini of target proteins to assemble signaling/transport complexes at the plasma membrane. These target protein partners include channels and transporters, such as NHE3, NBC3, and the transient receptor potential channels (TRPC4 and TRPC5), the sodium-phosphate cotransporter (Na + -Pi), and receptors, such as P2Y1 and β-adrenergic (Fanning and Anderson 1999;Shenolikar and Weinman 2001;Yun 2003;Shenolikar et al 2004;Fam et al 2005;Lee-Kwon et al 2005;. NHERFs also serve to recruit signaling molecules, such as PKA, PKC, and PI-3 kinase, to the receptor complexes (Fanning and Anderson 1999;Shenolikar and Weinman 2001;Yun 2003;Shenolikar et al 2004;Lee-Kwon et al 2005;Weinman et al 2005).…”