Plasma membrane association of p63 Rho guanine nucleotide exchange factor (p63RhoGEF) is mediated by palmitoylation and is required for basal activity in cells.

Life Sciences Institute, University of Michigan, Ann Arbor, Michigan 48109-2216, USA.
Journal of Biological Chemistry (Impact Factor: 4.65). 08/2011; 286(39):34448-56. DOI: 10.1074/jbc.M111.273342
Source: PubMed

ABSTRACT Activation of G protein-coupled receptors at the cell surface leads to the activation or inhibition of intracellular effector enzymes, which include various Rho guanine nucleotide exchange factors (RhoGEFs). RhoGEFs activate small molecular weight GTPases at the plasma membrane (PM). Many of the known G protein-coupled receptor-regulated RhoGEFs are found in the cytoplasm of unstimulated cells, and PM recruitment is a critical aspect of their regulation. In contrast, p63RhoGEF, a Gα(q)-regulated RhoGEF, appears to be constitutively localized to the PM. The objective of this study was to determine the molecular basis for the localization of p63RhoGEF and the impact of its subcellular localization on its regulation by Gα(q). Herein, we show that the pleckstrin homology domain of p63RhoGEF is not involved in its PM targeting. Instead, a conserved string of cysteines (Cys-23/25/26) at the N terminus of the enzyme is palmitoylated and required for membrane localization and full basal activity in cells. Conversion of these residues to serine relocates p63RhoGEF from the PM to the cytoplasm, diminishes its basal activity, and eliminates palmitoylation. The activity of palmitoylation-deficient p63RhoGEF can be rescued by targeting to the PM by fusion with tandem phospholipase C-δ1 pleckstrin homology domains or by co-expression with wild-type Gα(q) but not with palmitoylation-deficient Gα(q). Our data suggest that p63RhoGEF is regulated chiefly through allosteric control by Gα(q), as opposed to other known Gα-regulated RhoGEFs, which are instead sequestered in the cytoplasm, perhaps because of their high basal activity.

  • [Show abstract] [Hide abstract]
    ABSTRACT: The heterotrimeric G protein Gαq is a central player in signal transduction, relaying signals from activated G-protein-coupled receptors (GPCRs) to effectors and other proteins to elicit changes in intracellular Ca(2+), the actin cytoskeleton, and gene transcription. Gαq functions at the intracellular surface of the plasma membrane, as do its best-characterized targets, phospholipase C-β, p63RhoGEF, and GPCR kinase 2 (GRK2). Recent insights into the structure and function of these signaling complexes reveal several recurring themes, including complex multivalent interactions between Gαq, its protein target, and the membrane, that are likely essential for allosteric control and maximum efficiency in signal transduction. Thus, the plasma membrane is not only a source of substrates but also a key player in the scaffolding of Gαq-dependent signaling pathways.
    Trends in Pharmacological Sciences 11/2013; · 9.25 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The p63RhoGEF and GEFT proteins are encoded by the same gene and both members of the Dbl family of guanine nucleotide exchange factors. These proteins can be activated by the heterotrimeric G-protein subunit Gαq. We show that p63RhoGEF is located at the plasma membrane, whereas GEFT is confined to the cytoplasm. Live-cell imaging studies yielded quantitative information on diffusion coefficients, association rates and encounter times of GEFT and p63RhoGEF. Calcium signaling was examined as a measure of the signal transmission, revealing more efficient signaling through the membrane-associated p63RhoGEF. A rapamycin dependent recruitment system was used to dynamically alter the subcellular location and concentration of GEFT, showing efficient signaling through GEFT only upon membrane recruitment. Together, our results show efficient signal transmission through membrane located effectors, and highlight a role for increased concentration rather than increased encounter times due to membrane localization in the Gαq mediated pathways to p63RhoGEF and PLCβ.
    Scientific Reports 07/2013; 3:2284. · 5.08 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Some G‑protein‑coupled receptors regulate biological processes via Gα12/13‑ or Gαq/11‑mediated stimulation of Rho guanine nucleotide exchange factors (RhoGEFs). p63RhoGEF is known to be specifically activated by Gαq/11 and mediates a major part of the acute response of vascular smooth muscle cells to Angiotensin II treatment. In order to gain information about the dynamics of receptor-mediated activation of p63RhoGEF we developed a FRET-based assay to study interactions between Gαq-CFP and Venus-p63RhoGEF in single living cells. Upon activation of histaminergic H1 or muscarinic M3 receptors a robust FRET signal occurred that allowed for the first time to analyze the kinetics of this interaction in detail. On- and offset kinetics of Gαq-p63RhoGEF interactions closely resembled the kinetics of Gαq‑activity. Analysis of the effect of RGS2 on the dynamics of Gαq-activity and their interaction with p63RhoGEF showed that RGS2 is able to accelerate both deactivation of Gαq proteins as well as dissociation of Gαq and p63RhoGEF to a similar extent. Furthermore we were able to detect activation-dependent FRET between RGS2 and p63RhoGEF and observed a reduced p63RhoGEF mediated downstream signaling in the presence of RGS2. In summary, these observations support the concept of a functional, activation-dependent p63RhoGEF-Gαq-RGS2 complex.
    Biochemical Journal 12/2013; · 4.65 Impact Factor