A Transgenic MMTV-Flippase Mouse Line for Molecular Engineering in Mammary Gland and Breast Cancer Mouse Models.

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.

Dlg3 trafficking and apical tight junction formation is regulated by nedd4 and nedd4-2 e3 ubiquitin ligases.

The 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 PDZ scaffold NHERF-2 interacts with mGluR5 and regulates receptor activity.

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 mGluRs1 and 5are 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.

Heterodimerization with beta2-adrenergic receptors promotes surface expression and functional activity of alpha1D-adrenergic receptors.

The alpha1D-adrenergic receptor (alpha1D-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 alpha1D-AR cell surface expression. Strikingly, these screens revealed only two receptors capable of inducing robust increases in the amount of alpha1D-AR at the cell surface: alpha1B-AR and beta2-AR. Confocal imaging confirmed that coexpression with beta2-AR resulted in translocation of alpha1D-AR from intracellular sites to the plasma membrane. Additionally, coimmunoprecipitation studies demonstrated that alpha1D-AR and beta2-AR specifically interact to form heterodimers when coexpressed in HEK-293 cells. Ligand binding studies revealed an increase in total alpha1D-AR binding sites upon coexpression with beta2-AR, but no apparent effect on the pharmacological properties of the receptors. In functional studies, coexpression with beta2-AR significantly enhanced the coupling of alpha1D-AR to norepinephrine-stimulated Ca2+ mobilization. Heterodimerization of beta2-AR with alpha1D-AR also conferred the ability of alpha1D-AR to cointernalize upon beta2-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 alpha1D-AR with other receptors is crucial for receptor surface expression and function and also shed light on a novel mechanism of cross talk between alpha1- and beta2-ARs that is mediated through heterodimerization and cross-internalization.

P2Y1 receptor signaling is controlled by interaction with the PDZ scaffold NHERF-2.

P2Y(1) purinergic receptors (P2Y(1)Rs) 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 P2Y(1)R carboxyl terminus against a recently created proteomic array of PDZ (PSD-95/Drosophila Discs large/ZO-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 P2Y(1)R and NHERF-2 associate in cells, allowing NHERF-2-mediated tethering of P2Y(1)R to key downstream effectors such as phospholipase Cbeta. Finally, we found that coexpression of P2Y(1)R with NHERF-2 in glial cells prolongs P2Y(1)R-mediated Ca(2+) signaling, whereas disruption of the P2Y(1)R-NHERF-2 interaction by point mutations attenuates the duration of P2Y(1)R-mediated Ca(2+) responses. These findings reveal that NHERF-2 is a key regulator of the cellular activity of P2Y(1)R and may therefore determine cell-specific differences in P2Y(1)R-mediated signaling.