Aminoglutethimide

ABSTRACT Suramin, a polysulfonated naphthylurea, is under investigation for the treatment of several cancers. It interferes with signal transduction through G^sub s^, G^sub i^, and G^sub o^, but structural and kinetic aspects of the molecular mechanism are not well understood. Here, we have investigated the influence of suramin on coupling of bovine rhodopsin to Gt, where G-protein activation and receptor structure can be monitored by spectroscopic in vitro assays. Gt fluorescence changes in response to rhodopsin-catalyzed nucleotide exchange reveal that suramin inhibits G^sub t^ activation by slowing down the rate of complex formation between metarhodopsin-II and G^sub t^. The metarhodopsin-I/-II photoproduct equilibrium, GTPase activity, and nucleotide uptake by G^sub t^ are unaffected. Attenuated total reflection Fourier transform infrared spectroscopy shows that the structure of rhodopsin, metarhodopsin-II, and the metarhodopsin-II G^sub t^ complex is also not altered. Instead, suramin dissociates G^sub t^ from disk membranes in the dark, whereas metarhodopsin-II G^sub t^ complexes are stable. Forster resonance energy transfer suggests a suramin-binding site near Trp^sup 207^ on the G^sub t(alpha)^, subunit (K^sub d^ ~0.5 (mu)M). The kinetic analyses and the structural data are consistent with a specific perturbation by suramin of the membrane attachment site on G^sub t(alpha)^. Disruption of membrane anchoring may contribute to some of the effects of suramin exerted on other G-proteins.

INTRODUCTION

Suramin, a hexasulfonated polyaromatic naphthylurea (1.4 kDa), is under study for therapeutic activity in phase II trials in the treatment of several cancers (Mirza et al., 1997; Dawson et al., 1998; Dreicer et al., 1999). It exhibits anti-- angiogenetic and antiproliferative activity (Firsching et al., 1995) by interfering with the binding of several growth factors to their receptors (Coffey et al., 1987). These properties can be enhanced in suramin analogs (Gagliardi et al., 1998). However, adverse effects of suramin are dose-limiting (Chaudhry et al., 1996), and the molecular basis of its action is not well understood. The function of several cellular signaling proteins, such as protein-tyrosine phosphatases (Zhang et al., 1998) and protein kinase C (Khaled et al., 1995) is perturbed, and recent interest in suramin focuses on its ability to interfere also with signaling through G-protein-coupled receptors (GPCRs). GPCRs are heptahelical transmembrane proteins that respond to extracellular signals, such as binding of a hormone or a neurotransmitter, by catalyzing GDP/GTP exchange in cytosolic guanosine-- nucleotide-binding proteins (G-proteins) (for reviews see Ji et al., 1998; Gether, 2000). Suramin has been shown to uncouple alpha^sub 2^- and beta^sub 2^-adrenergic receptors from G^sub i^ and G^sub s^, respectively (Huang et al., 1990). It has also been shown that suramin inhibits activation of pertussis-toxin-sensitive G-proteins by delta-opioid receptors in NG 108-15 cell membranes, whereas nucleotide exchange induced by serum factors binding to an unidentified receptor was not affected (Butler et al., 1988). Based on these initial observations, the potential of suramin analogs to interfere with signaling by different receptor G-protein tandems has been investigated systematically. A suramin analog has been described that uncouples A^sub 1^ adenosine and D^sub 2^ dopamine receptors from G^sub i^/G^sub o^ with different specificity (Beindl et al., 1996; Waldhoer et al., 1998), and a number of analogs have been synthesized that act as subtype-specific G-protein inhibitors (Hohenegger et al., 1998; Waldhoer et al., 1998). Such studies emphasize the role of G-proteins as potential drug targets (Holler et al., 1999). However, details of the molecular mechanism by which suramin affects structural and kinetic parameters of receptor G-protein interactions remain to be elucidated. In an attempt to exploit a well characterized in vitro model system for GPCR signaling, we have investigated the effect of suramin on activation of transducin (G^sub t^) by the bovine photoreceptor rhodopsin. For this system, biophysical assays for conformational changes of the receptor, G^sub t^ binding, and G^sub t^ activation are available. Based on the homology of class I (rhodopsin-like) GPCRs, a study of the action of suramin on rhodopsin G^sub t^ interactions helps to identify molecular mechanisms by which the drug may affect signaling in related systems.

Rhodopsin is a prototypical GPCR (Helmreich and Hofmann, 1996; Menon et al., 2001; Okada et al., 2001) and the only one for which a crystal structure has been solved (Palczewski et al., 2000). Rhodopsin is unique as it is not ligand-activated. Instead, 11-cis-retinal is covalently attached to Lys^sup 296^ of the apoprotein via a protonated Schiff base (Oseroff and Callender, 1974). Photoisomerization to all-trans-retinal promotes structural changes involving helix-helix interactions and rigid body movements (Farrens et al., 1996; Han et al., 1996; Sheikh et al., 1996). As a consequence, cytosolic domains of the active metarhodopsin-II (MII) state bind G^sub t^ and catalyze GDP/GTP exchange. Signaling by the rhodopsin G^sub t^ tandem has been optimized for maximal light sensitivity, which implies minimization of dark noise (Birge and Barlow, 1995; Rieke and Baylor, 1996). Correspondingly, basal nucleotide exchange, typical of other G-proteins, is negligible in G^sub t^. The unique features of rhodopsin and G^sub t^ suggest that interference with receptor coupling is the predominant mode of action by which a pharmacologically active substance may modulate G^sub t^ activation. Other potential perturbations such as interference with basal G-protein activation or competition with agonist binding are not expected to contribute to the readout from this model system. We have applied fluorescence spectroscopy to analyze binding of suramin to G^sub t(alpha)^ and to monitor rhodopsin-catalyzed G^sub t^ activation. By attenuated total reflection (ATR) Fourier transform infrared (FTIR) spectroscopy we have investigated the influence of suramin on the structure of rhodopsin and the MIIG^sub t^ complex. We show that suramin binds to G^sub t(alpha)^ but does not affect either GTP uptake or GTPase activity. Likewise, structural changes during rhodopsin activation are not influenced. Instead, the rate of MIIG^sub t^ formation is reduced by a dose-dependent inhibition of membrane anchoring of Gt. In addition to inferences on receptor G-protein coupling in nonvisual signaling, the data are relevant for the molecular characterization of side effects on vision in patients receiving suramin treatment. A variety of ocular symptoms have been described (for example, Hemady et al., 1996).

MATERIALS AND METHODS

Purification of rhodopsin and transducin

This research was supported by Deutsche Forschungsgemeinschaft grant FA 248/4-1 to K.F.

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Nicole Lehmann,* Gopala Krishna Aradhyam,^ and Karim Fahmy*

*Institut fOr Molekulare Medizin and Zellforschung, Albert-Ludwigs-Universitat, D-79104 Freiburg, Germany; and ^Howard Hughes Medical Institute, Laboratory of Molecular Biology and Biochemistry, Rockefeller University, New York, New York 10021 USA

Submitted July 26, 2001, and accepted for publication October 1, 2001.

Address reprint requests to Dr. Karim Fahmy, Institut fair Molekulare Medizin and Zellforschung, Universitat Freiburg, Albertstrasse 23, D-79104 Freiburg, Germany. Tel.: 49-761-203-5380; Fax: 49-761-203-- 2921; E-mail: fahmy@uni-freiburg.de.

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