The Non-ABC Drug Transporter RLIP76 (RALBP-1) Plays a Major Role in the Mechanisms of Drug Resistance

Abstract

RLIP76 or Ral binding protein (RalBP-1) was initially cloned as a Ral-effector that was proposed as a link between Ral and Ras pathways. This protein is encoded in humans on chromosome 18p11.3 by a gene with 11 exons and 9 introns and is found ubiquitously from drosophila to humans. RLIP76 displays inhibitory GTPase activity toward Rho/Rac class G-protein cdc42 which is involved in regulation of cytoskeletal organization, lamellipodia, cell migration and apoptosis via Ras. We have recently shown that RLIP76 is also a multispecific transporter of chemotherapeutic agents and glutathione conjugates (GS-E). In human cells RLIP76 accounts for more than two third of the transport activity for GS-E and drugs as opposed to the ABC-transporters including MRP1, which account for less than one third of this activity. Evidence is mounting that RLIP76 is a stress-responsive multi-specific, non-ABC transporter which represents an entirely novel link between stress-inducible G-protein signaling, receptor tyrosine-kinase signaling, endocytosis, heat-shock and stress defense pathways, and transport mediated drug-resistance. The expression of RLIP76 is significantly greater in human cancer cells of diverse origin as compared to the non-malignant cells. Inhibition of RLIP76, using antibodies towards a cell surface epitope, or depletion of RLIP76 using either siRNA or anti-sense phosphorothioate oligonucleotides preferentially causes apoptosis in malignant cells. Administration of RLIP76 antibodies, siRNA, or anti-sense oligonucleotides to mice bearing syngeneic B16 mouse melanoma tumors causes rapid and complete regression of tumors. Studies summarized in this review strongly suggest that RLIP76 is a logical target for clinical intervention of not only multi-drug resistance but also for diseases resulting from oxidative stress.

Physiological and Pharmacological Significance of Glutathione-Conjugate Transport

“…Sufficient quantities of bacterially expressed RLIP76 have allowed detailed studies on the role of RLIP76 in the transport of GSH conjugates of xeno-and endobiotics (Awasthi et al, 2000 and its contribution to the mechanisms underrlying drug resistance of cancer cells . Furthermore, these studies provided clues for a link between GS-E transport and cell cycle regulation because RLIP76 was shown to be the major determinant of cellular concentrations of 4-HNE, a key second-messenger molecule involved in either pro-apoptotic signaling or proliferation depending upon its concentration in cells (Sharma et al, 2004;Awasthi et al, 2007). These findings as elaborated later in this review provide novel directions to approaches to cancer chemotherapy.…”

An overview of cancer multidrug resistance: a still unsolved problem

“…In vitro studies have provided evidence that cellular transporter proteins other than ABC-transporters may also contribute to anticancer drug resistance. Thus, RALBP1 (RLIP76), a 76 kDa Ral-binding, Rho/Rac-GAP and Ral effector protein can act as a transporter of structurally unrelated amphiphilic chemotherapeutic drugs such as A C H T U N G T R E N N U N G doxorubicin and glutathione-electrophile conjugates [98][99][100]. Analogous to ABCtransporters, RALBP1 mediates transport of xenobiotics in an ATP-dependent manner, but it has no sequence homology with the ABC-transporter family and differs from the ABC-transporters in several other important aspects.…”

1H, 13C and 15N resonance assignments for the active conformation of the small G protein RalB in complex with its effector RLIP76

“…RLIP76 is implicated in endocytosis though its interactions with POB1 and the l2 subunit of the AP2 complex (Yamaguchi et al 1997;Ikeda et al 1998;Jullien-Flores et al 2000). A role for RLIP76 in the efflux of xenobiotic substances has also been investigated and is thought to be dependent on the ability of RLIP76 to bind ATP and therefore act as a pump (Awasthi et al 2007). Here we report the backbone and sidechain assignments for RalB bound to the nonhydrolysable analogue GMPPNP and the Ral binding domain of RLIP76 (residues 393-446).…”