Effects of Differential Sulfhydryl Group-Specific Labeling on the Rhodopsin and Guanine Nucleotide Binding Activities of Transducin Julio O. Ortiz and Jose ´ Bubis 1 Departamento de Biologı ´a Celular, Universidad Simo ´n Bolı´var, Apartado 89.000, Valle de Sartenejas, Caracas 1081-A, Venezuela Received May 19, 2000, and in revised form November 16, 2000; published online February 19, 2001 The role of transducin sulfhydryl groups was ex- amined by chemical modification with four different reagents: 4-acetamido-4-maleimidyl-stilbene-2, 2 disulfonic acid (AMDA); 4-vinyl pyridine (VP); 2-ni- tro-5-thiocyano benzoic acid (NTCBA); and 2, 5-dime- thoxystilbene-4-maleimide (DM). All these com- pounds rapidly inhibited the [ 3 H]GMPpNp-binding activity of transducin stimulated by photoexcited rhodopsin (R*). Sedimentation experiments showed that the labeling of transducin with AMDA or VP hindered its binding to R* while NTCBA-modified transducin was capable of interacting with the pho- toreceptor protein. In contrast, DM-labeled transdu- cin precipitated even in the absence of R * . Photoac- tivated rhodopsin was capable of protecting against the observed AMDA and NTCBA inhibition in trans- ducin function, but not against the inactivation caused by VP or DM. These results suggest the exis- tence of different functional cysteines on transducin that are located in the proximity of the interaction site with the photoreceptor protein, near the gua- nine nucleotide binding site, or in regions involved in the structural changes taking place upon protein activation. With the use of these reagents, transdu- cin appears to be “frozen” in various conformational stages of its cycle, providing conditions for studying two of the initial steps of the visual process: the light-dependent binding of transducin to rhodopsin and the transducin guanine nucleotide exchange re- action. © 2001 Academic Press Key Words: transducin; cysteine labeling; rhodopsin; vision; G proteins. Activation of heterotrimeric guanine nucleotide binding proteins (G proteins), 2 via stimulation of hep- tahelical receptors by sensory, hormonal, or neuro- transmitter signals, results in the regulation of a vari- ety of downstream enzymes and ion channels (1–3). Thousands of G protein-coupled receptors have been identified, which share a common architecture contain- ing seven transmembrane-spanning segments (4). G proteins are composed of -, -, and -subunits, acting as two functional units, G  and G  , and comprise a highly heterogeneous family of proteins that includes at least 20 distinct G  ,6G  , and 12 G  isoforms (1, 5, 6), allowing many combinatorial possibilities. The G protein cycle is initiated when an extracellu- lar signal activates a cell surface receptor. The acti- vated receptor interacts with the inactive G protein heterotrimer, catalyzing the release of GDP from the G protein -subunit, and giving rise to an empty nucle- otide-binding pocket in this polypeptide. The G  sub- unit and the G  unit remain bound to the receptor in a tight complex, until GTP binds to the empty -sub- unit. Then GTP induces conformational changes in G  , which consequently activate the G protein, causing G  -GTP to dissociate from G  and both G  -GTP and 1 To whom correspondence and reprint requests should be ad- dressed. Fax: (58-2) 906-3064. E-mail: jbubis@usb.ve. 2 Abbreviations used: AMDA, 4-acetamido-4'-maleimidyl- stilbene-2, 2'-disulfonic acid; BCIP, 5-bromo-4-chloro-3-indolyl phos- phate; DM, 2, 5-dimethoxystilbene-4'-maleimide; DMF, dimethylfor- mamide; DTT, dithiothreitol; GMPpNp, ,-imido-guanosine 5'- triphosphate; G proteins, heterotrimeric guanine nucleotide-binding proteins; HPLC, high-performance liquid chromatography; IAA, io- doacetic acid; NTCBA, 2-nitro-5-thiocyano benzoic acid; NBT, nitro blue tetrazolium; R*, photoexcited rhodopsin; ROS, rod outer seg- ments; SDS–PAGE, SDS–polyacrylamide gel electrophoresis; T or T  , transducin; T AMDA , AMDA-modified transducin; T DM , DM-modi- fied transducin; T NTCBA , NTCBA-modified transducin; T VP , VP-modi- fied transducin; T:R*, transducin-activated rhodopsin complex; TPCK, tosylphenyl alanyl chloromethyl ketone; VP, 4-vinyl pyridine. 0003-9861/01 $35.00 233 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved. Archives of Biochemistry and Biophysics Vol. 387, No. 2, March 15, pp. 233–242, 2001 doi:10.1006/abbi.2000.2219, available online at http://www.idealibrary.com on