Intracellular localization of the P21rho proteins.

Abstract

Abstract. The three mammalian ras proteins associate specifically with the plasma membrane and this is essential for their biological activity. Two signals encoded within the extreme COOH terminus of the proteins specify this cellular localization; a CAAX box in combination with either a polybasic domain (p21K-~B) or a palmitoylation site (p21 m-~ and p21N-~). All members of the ras-like and rho-like subfamilies of the ras superfamily of small GTP-binding proteins also have CAAX boxes with potential second site sequences resembling either p21K-~B or P21N-~m-~. However it is not at all clear that they are each located at the plasma membrane, and in fact one of the ras-like proteins, rapl, has been localized to the Golgi (Beranger et al., 1991). None of the mammalian rho-like subfamily has yet been localized. Three forms (A, B, and C) of p21 ~h~ the prototype of this family are known; the COOH termini of p21 rh~ and p21 rh~ resemble p21K-'~B with a polybasic domain, whereas p21 rh~ resembles p21N-r~/m-r~ with two cysteine residues as potential palmitoylation sites. Despite this similarity to the p21 ~ proteins, rho proteins have been purified from both particulate and cytosolic fractions of a variety of tissues. In order to localize definitively the three rho proteins we have used an epitope tagging approach coupled to microinjection of living cells. We show that a small fraction of all three proteins is localized to the plasma membrane but the majority of p21 rh~ and p21 rh~ is cytosolic whereas p21 ~h~ is associated with early endosomes and a pre-lysosomal compartment. Along with the results obtained with chimeric molecules using heterologous proteins attached to rho COOH termini, this suggests that the p2P h~ proteins cycle on and off the plasma membrane and this may have important implications for their biological function.

RhoA/ROCK-mediated switching between Cdc42- and Rac1-dependent protrusion in MTLn3 carcinoma cells

“…Anti-Rho staining was also observed in numerous punctate structures, which could result from RhoB in endosomal structures. These data are consistent with previous observations of a small fraction of RhoA in the plasma membrane [35]. Anti-Rho staining was specific, as it was abolished by pre-absorption with GST-RhoA (Fig 1C) but not GST (data not shown).…”

“…At 1 min, Rho was localized to a narrow band that extended approximately 0.5 μm into the cell (Fig 1B middle panel), which is the zone of f-actin accumulation in EGF-stimulated MTLn3 cells [34]. We presume that this is staining pattern is largely due to RhoA, since RhoB is confined to endosomes [35]. However, by 3 minutes, anti-Rho staining is localized to a broader region that began approximately 0.5 μm behind the leading edge and extended 2 μm into the cell (Fig.…”

ROCK I-mediated activation of NF-κB by RhoB

“…It also has a unique pattern of cellular distribution. It has been localized to specialized membrane compartments including the Golgi, endosomal vesicles, and the multivesicular body (MVB) [3,4]. This localization is consistent with several studies which suggest an important role for RhoB in endosome-mediated trafficking of growth factor receptors [4,5].…”

A mutant form of the rho protein can restore stress fibers and adhesion plaques in v-src transformed fibroblasts

“…The next point we investigated was the intracellular localization of the transfected rho. Hall and coworkers have reported that rho proteins can be detected at the plasma membrane of transfected cells, but most of these proteins are localized in the cytosol (Adamson et al, 1992). From these data it was concluded that the rho proteins need to undergo a membrane/cytosol cycle to be functional.…”

“…In the mammalian cell, only a fraction of the rho proteins can be detected on the lipid bilayer of the plasma membrane (Adamson et al, 1992). Posttranslational isoprenylation of ras-proteins is the ®rst step in the translocation process.…”