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...

The ␣ 1D -adrenergic receptor (␣ 1D -AR) is a G protein-coupled receptor (GPCR) that is poorly trafficked to the cell surface and largely nonfunctional when heterologously expressed by itself in a variety of cell types. We screened a library of approximately 30 other group I GPCRs in a quantitative luminometer assay for the ability to promote ␣ 1D -AR cell surface expression. Strikingly, these screens revealed only two receptors capable of inducing robust increases in the amount of ␣ 1D -AR at the cell surface: ␣ 1B -AR and ␤ 2 -AR. Confocal imaging confirmed that coexpression with ␤ 2 -AR resulted in translocation of ␣ 1D -AR from intracellular sites to the plasma membrane. Additionally, coimmunoprecipitation studies demonstrated that ␣ 1D -AR and ␤ 2 -AR specifically interact to form heterodimers when coexpressed in HEK-293 cells. Ligand binding studies revealed an increase in total ␣ 1D -AR binding sites upon coexpression with ␤ 2 -AR, but no apparent effect on the pharmacological properties of the receptors. In functional studies, coexpression with ␤ 2 -AR significantly enhanced the coupling of ␣ 1D -AR to norepinephrine-stimulated Ca 2ϩ mobilization. Heterodimerization of ␤ 2 -AR with ␣ 1D -AR also conferred the ability of ␣ 1D -AR to cointernalize upon ␤ 2 -AR agonist stimulation, revealing a novel mechanism by which these different adrenergic receptor subtypes may regulate each other's activity. These findings demonstrate that the selective association of ␣ 1D -AR with other receptors is crucial for receptor surface expression and function and also shed light on a novel mechanism of cross talk between ␣ 1 -and ␤ 2 -ARs that is mediated through heterodimerization and cross-internalization.

SummaryThe Drosophila Discs large (Dlg) scaffolding protein acts as a tumor suppressor regulating basolateral epithelial polarity and proliferation. In mammals, four Dlg homologs have been identified; however, their functions in cell polarity remain poorly understood. Here, we demonstrate that the X-linked mental retardation gene product Dlg3 contributes to apical-basal polarity and epithelial junction formation in mouse organizer tissues, as well as to planar cell polarity in the inner ear. We purified complexes associated with Dlg3 in polarized epithelial cells, including proteins regulating directed trafficking and tight junction formation. Remarkably, of the four Dlg family members, Dlg3 exerts a distinct function by recruiting the ubiquitin ligases Nedd4 and Nedd4-2 through its PPxY motifs. We found that these interactions are required for Dlg3 monoubiquitination, apical membrane recruitment, and tight junction consolidation. Our findings reveal an unexpected evolutionary diversification of the vertebrate Dlg family in basolateral epithelium formation.

The two members of the group I metabotropic glutamate receptor family, mGluR1 and mGluR5, both couple to G q to mediate rises in intracellular calcium. The alternatively spliced C termini (CT) of mGluRs 1 & 5 are known to be critical for regulating receptor activity and to terminate in motifs suggestive of potential interactions with PDZ domains. We therefore screened the CTs of both mGluR1a and mGluR5 against a PDZ domain proteomic array. Out of 96 PDZ domains examined, the domain that bound most strongly to mGluR5-CT was the second PDZ domain of the Na ؉ /H ؉ exchanger regulatory factor 2 (NHERF-2). This interaction was confirmed by reverse overlay, and a single point mutation to the mGluR5-CT was found to completely disrupt the interaction. Full-length mGluR5 robustly associated with full-length NHERF-2 in cells, as assessed by co-immunoprecipitation and confocal microscopy experiments. In contrast, mGluR1a was found to bind NHERF-2 in vitro with a weaker affinity than mGluR5, and furthermore mGluR1a did not detectably associate with NHERF-2 in a cellular context. Immunohistochemical experiments revealed that NHERF-2 and mGluR5 exhibit overlapping patterns of expression in mouse brain, being found most abundantly in astrocytic processes and postsynaptic neuronal elements. In functional experiments, the interaction of NHERF-2 with mGluR5 in cells was found to prolong mGluR5-mediated calcium mobilization and to also potentiate mGluR5-mediated cell death, whereas coexpression of mGluR1a with NHERF-2 had no evident effects on mGluR1a functional activity. These observations reveal that NHERF-2 can selectively modulate mGluR5 signaling, which may contribute to cell-specific regulation of mGluR5 activity.Many of the physiological effects of L-glutamate as a neurotransmitter are mediated via stimulation of metabotropic glutamate receptors (mGluRs), 2 which are considered to be important therapeutic targets in the potential treatment of a number of different diseases (1-3). The mGluRs are G protein-coupled receptors that regulate the physiology of neurons and glia through activation of various secondary messenger systems (4). Eight subtypes of mGluRs have been identified and classified into three groups (I, II, III) based on sequence homology, signaling pathways and pharmacological selectivity. The various mGluR subtypes are known to display differential regional and cellular distributions throughout the mammalian brain (4). Activation of group I mGluRs (mGluR1 and mGluR5) leads to signal transduction primarily through coupling to G q and phospholipase C, which then hydrolyzes phosphatidylinositol 4,5-bisphosphate (PIP 2 ) to form the second messenger inositol 1,4,5-phosphate (IP 3 ). IP 3 binding to its receptor in the membrane of the endoplasmic reticulum causes release of free Ca 2ϩ in the cytosol, which generates a signaling cascade. Group I mGluRs can also couple to other signaling pathways in a cell type-specific manner (5). Despite many similarities in their signaling pathways, mGluR1 and mGluR5 expressed in th...

Genetically engineered mouse models have become an indispensable tool for breast cancer research. Combination of multiple site-specific recombination systems such as Cre/loxP and Flippase (Flp)/Frt allows for engineering of sophisticated, multi-layered conditional mouse models. Here, we report the generation and characterization of a novel transgenic mouse line expressing a mouse codon-optimized Flp under the control of the mouse mammary tumor virus (MMTV) promoter. These mice show robust Flp-mediated recombination in luminal mammary gland and breast cancer cells but no Flp activity in non-mammary tissues, with the exception of limited activity in salivary glands. These mice provide a unique tool for studying mammary gland biology and carcinogenesis in mice.