The neuropeptides bombesin and endothelin-1 stimulate prostate cancer (PC) cell migration and invasion (J Clin Invest, 2000; 106: 1399–1407). The intracellular signaling pathways that direct this cell movement are not well delineated. The monomeric GTPase RhoA is required for migration in several cell types including neutrophils, monocytes and fibroblasts. We demonstrate that bombesin-stimulated PC cell migration occurs via the heterotrimeric G-protein-coupled receptors (G-protein) Gα13 subunit leading to activation of RhoA, and Rho-associated coiled-coil forming protein kinase (ROCK). Using siRNA to suppress expression of the three known G-protein α-subunit-associated RhoA guanine nucleotide exchange factors (GEFs), we also show that two of these RhoA GEFs, PDZ-RhoGEF and leukemia-associated RhoGEF (LARG), link bombesin receptors to RhoA in a non-redundant manner in PC cells. We next show that focal adhesion kinase, which activates PDZ-RhoGEF and LARG, is required for bombesin-stimulated RhoA activation. Neutral endopeptidase (NEP) is expressed on normal prostate epithelium whereas loss of NEP expression contributes to PC progression. We also demonstrate that NEP inhibits neuropeptide activation of RhoA. Together, these results establish a contiguous signaling pathway from the bombesin receptor to ROCK in PC cells, and they implicate NEP as a major regulator of neuropeptide-stimulated RhoA in these cells. This work also identifies members of this signaling pathway as potential targets for rational pharmacologic manipulation of neuropeptide-stimulated migration of PC cells.

The physiological properties of most ion channels are defined experimentally by functional expression of their pore-forming α subunits in Xenopus laevis oocytes. Here, we cloned a family ofXenopus KCNE genes that encode MinK-related peptide K+ channel β subunits (xMiRPs) and demonstrated their constitutive expression in oocytes. Electrophysiological analysis of xMiRP2 revealed that when overexpressed this gene modulates human cardiac K+ channel α subunits HERG (human ether-a-go-go-related gene) and KCNQ1 by suppressing HERG currents and removing the voltage dependence of KCNQ1 activation. The ability of endogenous levels of xMiRP2 to contribute to the biophysical attributes of overexpressed mammalian K+ channels in oocyte studies was assessed next. Injection of an xMiRP2 sequence-specific short interfering RNA (siRNA) oligo reduced endogenous xMiRP2 expression 5-fold, whereas a control siRNA oligo had no effect, indicating the effectiveness of the RNA interference technique in Xenopus oocytes. The functional effects of endogenous xMiRP2 silencing were tested using electrophysiological analysis of heterologously expressed HERG channels. The RNA interference-mediated reduction of endogenous xMiRP2 expression increased macroscopic HERG current as much as 10-fold depending on HERG cRNA concentration. The functional effects of human MiRP1 (hMiRP1)/HERG interaction were also affected by endogenous xMiRP2. At high HERG channel density, at which the effects of endogenous xMiRP2 are minimal, hMiRP1 reduced HERG current. At low HERG current density, hMiRP1 paradoxically up-regulated HERG current, a result consistent with hMiRP1 rescuing HERG from suppression by endogenous xMiRP2. Thus, endogenous Xenopus MiRP subunits contribute to the base-line properties of K+ channels like HERG in oocyte expression studies, which could explain expression level- and expression system-dependent variation in K+channel function.

Rho GTPases control fundamental cellular processes, including cytoskeletal reorganization and transcription. Rho guanine nucleotide exchange factors (GEFs) compose a large (>65) and diverse family of related proteins that activate Rho GTPases. Lsc/p115-RhoGEF is a Rho-specific GEF required for normal B and T lymphocyte function. Despite its essential role in lymphocytes, Lsc/p115-RhoGEF signaling in vivo is not well understood. To define Lsc/p115-RhoGEF signaling pathways in vivo, we set out to identify proteins that interact with regulatory regions of Lsc. The 146-amino acid C terminus of Lsc contains a predicted coiled-coil domain, and we demonstrated that deletion of this C terminus confers a gain of function in vivo. Surprisingly, a yeast two-hybrid screen for proteins that interact with this regulatory C terminus isolated a larger C-terminal fragment of Lsc itself. Co-immunoprecipitation experiments in mammalian cells demonstrated that Lsc specifically homo-oligomerizes and that the coiled-coil domain in the C terminus is required for homo-oligomerization. Mutagenesis experiments revealed that homo-oligomerization and negative regulation are distinct functions of the C terminus. Two novel isoforms of Lsc found in the spleen lack portions of this C terminus, including the coiled-coil domain. Importantly, the C termini of both isoforms confer a gain of function and eliminate homo-oligomerization. These results define two important features of Lsc signaling. First, Lsc homo-oligomerizes and is negatively regulated through domains in its C terminus; and second, functionally distinct isoforms of Lsc lacking these domains are present in the spleen.

Delayed rectifier potassium current diversity and regulation are essential for signal processing and integration in neuronal circuits. Here, we investigated a neuronal role for MinK-related peptides (MiRPs), membrane-spanning modulatory subunits that generate phenotypic diversity in cardiac potassium channels. Native coimmunoprecipitation from rat brain membranes identified two novel potassium channel complexes, MiRP2-Kv2.1 and MiRP2-Kv3.1b. MiRP2 reduces the current density of both channels, slows Kv3.1b activation, and slows both activation and deactivation of Kv2.1. Altering native MiRP2 expression levels by RNAi gene silencing or cDNA transfection toggles the magnitude and kinetics of endogenous delayed rectifier currents in PC12 cells and hippocampal neurons. Computer simulations predict that the slower gating of Kv3.1b in complexes with MiRP2 will broaden action potentials and lower sustainable firing frequency. Thus, MiRP2, unlike other known neuronal beta subunits, provides a mechanism for influence over multiple delayed rectifier potassium currents in mammalian CNS via modulation of alpha subunits from structurally and kinetically distinct subfamilies.

In low flow states, underestimation errors occur when the Gorlin formula is used to calculate valve area. A model of valvular stenosis designed to examine changes in the hydraulic discharge coefficient (Cd) and coefficient of orifice contraction (Cc) may explain these errors. Unsteady flow was examined in a pulsatile pump model and in a dog model. Valve areas were calculated from pressure and flow data using: a modified form of the Gorlin formula (assuming constant values for Cd and Cc) and a corrected formula (with values of Cd and Cc obtained from steady state data). Valve area was also calculated using the continuity equation with velocity and flow data (constant Cc). Flow velocities were measured using a newly designed ultrasound Doppler catheter capable of resolving flow velocities of up to 5.5 m/s. Both the corrected formula and continuity equation were highly predictive of actual valve area (r = 0.99, slope or M = 0.96 and r = 0.99, M = 1.06, respectively). The modified Gorlin equation was less accurate and tended to underestimate valve areas (r = 0.87, M = 0.83). This underestimation was most notable at low rates of flow (Gorlin: r = 0.94, M = 0.53; continuity: r = 0.93, M = 0.81 and r = 0.94, M = 0.89, respectively) more accurately than the modified Gorlin formula (r = 0.69, M = 0.49). In patients with low cardiac output, hemodynamic formulas, such as the Gorlin formula, which assume a constant value for the hydraulic discharge coefficient (Cd), may be less accurate than formulas using either a corrected value of Cd or Doppler-determined flow velocity and mean systolic flow.