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Efflux of cytosolic Ca[sup2+] mediated by plasma membrane Ca[sup2+]-ATPases (PMCA) plays a key role in fine tuning the magnitude and duration of Ca[sup2+] signaling following activation of G-protein-coupled receptors. However, the molecular mechanisms that underpin the trafficking of PMCA to the membrane during Ca[sup2+] signaling remain largely unexplored in native cell models. One potential mechanism for the recruitment of proteins to the plasma membrane involves PDZ interactions. In this context, we investigated the role ofPMCAinteractions with the Na[sup+]/H[sup+] exchanger regulatory factor 2 (NHERF-2) during muscarinicinduced Ca[sup2+] mobilization in the HT-29 epithelial cell line. GST pull-downs in HT-29 cell lysates showed that the PDZ2 module of NHERF-2 bound to the PDZ binding motif on the C terminus of PMCA. Co-immunoprecipitations confirmed that PMCA1b and NHERF-2 associated under normal conditions in HT-29 cells. Cell surface biotinylations revealed significant increases in membrane-associated NHERF-2 and PMCA within 60 s following muscarinic activation, accompanied by increased association of the two proteins as seen by confocal microscopy. The recruitment of NHERF-2 to the membrane preceded that of PMCA, suggesting that NHERF-2 was involved in nucleating an efflux complex at the membrane. The muscarinic-mediated translocation of PMCA was abolished when NHERF-2 was silenced, and the rate of relative Ca[sup2+] efflux was also reduced. These experiments also uncovered a NHERF-2-independent PMCA retrieval mechanism. Our findings describe rapid agonist-induced translocation of PMCA in a native cell model and suggest that NHERF-2 plays a key role in scaffolding and maintaining PMCA at the cell membrane. [ABSTRACT FROM AUTHOR]

Lysophosphatidic acid (LPA) is a mediator of multiple cellular responses. LPA mediates its effects predominantly through the G protein-coupled receptors LPA₁, LPA₂, and LPA₃. In the present work, we studied LPA₂-mediated signaling using human colon cancer cell lines, which predominantly express LPA₂. LPA₂ activated Akt and Erk1/2 in response to LPA. LPA mediated Akt activation was inhibited by pertussis toxin (PTX), whereas Erkl/2 activation was completely inhibited by a blocker of phospholipase Cβ, U-73122. LPA also induced interleukin-8 (IL-8) synthesis in the colon cancer cells by primarily activating LPA₂ receptor. We also found that LPA₂ interacts with Na+/H+ exchanger regulatory factor 2 (NHERF2). Activation of Akt and Erkl/2 was significantly attenuated by silencing of NHERF2 expression by RNA interference, suggesting a pivotal role of NHERF2 in LPA₂-mediated signaling. We found that expression of LPA₂ was elevated, whereas expression of LPA₁ downregulated in several types of cancers, including ovarian and colon cancer. We conclude that LPA₂ is the major LPA receptor in colon cancer cells and cellular signals by LPA₂ are largely mediated through its ability to interact with NHERF2. [ABSTRACT FROM AUTHOR]

The intestinal epithelium interacts dynamically with the immune system to maintain its barrier function to protect the host, while performing the physiological roles in absorption of nutrients, electrolytes, water and minerals. The importance of lysophosphatidic acid (LPA) and its receptors in the gut has been progressively appreciated. LPA signaling modulates cell proliferation, invasion, adhesion, angiogenesis, and survival that can promote cancer growth and metastasis. These effects are equally important for the maintenance of the epithelial barrier in the gut, which forms the first line of defense against the milieu of potentially pathogenic stimuli. This review focuses on the LPA-mediated signaling that potentially contributes to inflammation and tumor formation in the gastrointestinal tract. [ABSTRACT FROM AUTHOR]

The two members of the group I metabotropic glutamate receptor family, mGluR1 and mGluR5, both couple to Gq 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 co- expression 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. [ABSTRACT FROM AUTHOR]