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Abstract: Neurotensin receptors (NT-R) and the epidermal growth factor receptors (EGF-R) are commonly overexpressed in many epithelial origin tumours. In addition to their role as mitogenic mediators through specific cell signalling, recent studies indicate that the activity/expression of scaffold proteins responsible for the assembly and coordination of the signalling complexes may also have central roles in epithelial transformation. In particular, the “epithelial” PSD-95/Dlg/Zo-1 (PDZ) scaffold/adapter protein, Na+/H+ exchanger regulatory factor isoform one (NHERF-1), has been identified as a potential regulator of cellular transformation. NHERF-1 is a known regulator of EGF-R function and plays numerous roles in G-protein-coupled receptor signalling. Because of the synergistic signalling between these two potent mitogens, we investigated a potential role for NHERF-1 in the molecular mechanism linking the aberrant proliferative phenotype initiated by some G-Protein-coupled receptor activators in the colon adenocarcinoma HT-29 cell line. Knockdown (80%) of endogenous NHERF-1 leads to significant reduction in proliferation rate; an effect that could not be recovered by exogenous application of either NT or EGF. Inhibition of the EGF-R with AG1487 also inhibited proliferation and this effect could not be recovered with NT. Knockdown of NHERF-1 significantly altered the expression of the EGF-R, and almost completely abolished the NT-mediated increases in intracellular free Ca2+. Knockdown of NHERF-1 also attenuated UTP-mediated purinergic Ca2+ signalling. Taken together, these data suggest that NHERF-1 plays a more central role in cell proliferation by modulating Gq-mediated signalling pathways. [Copyright &y& Elsevier]

Abstract: Using the whole-cell patch-clamp technique, we identified an amiloride (AMI)-sensitive Na+ current in cystic fibrosis cells, JME/CF15, growing in standard medium. The reversal potential of this current depended on Na+ concentrations and the cation selectivity was much higher for Na+ than for K+, indicating that the current is through ENaC channels. In contrast, cells from EGF-containing medium lacked AMI-sensitive Na+ currents. In permeabilized cells growing in EGF-containing medium, αENaC was mainly detected in a perinuclear region, while in cells from standard medium it was distributed over the cell body. Western-blot analysis showed that in standard medium cells expressed fast-migrating EndoH-insensitive and slow-migrating EndoH-sensitive αENaC fractions, while in cells growing in the presence of EGF, αENaC was only detected as the fast-migrating EndoH-insensitive fraction. Long-term incubation of cells with EGF resulted in an increased basal Ca2+ level, [Ca2+]i. A similar increase of [Ca2+]i was also observed in the presence of 2μM thapsigargin, resulting in inhibition of ENaC function. Thus, in JME/CF15 cells inhibition of the ENaC function by chronic incubation with EGF is a Ca2+-mediated process that affects trafficking and surface expression of ENaC channels. [Copyright &y& Elsevier]