Proc. Nati. Acad. Sci. USA Vol. 86, pp. 6878-6882, September 1989 Biochemistry Three cytoplasmic loops of rhodopsin interact with transducin (vision/receptor/guanine nucleotide-binding protein/signal transduction/competing peptide) B. KONIG*, A. ARENDTt, J. H. MCDOWELLt, M. KAHLERT*, P. A. HARGRAVEtt, AND K. P. HOFMANN* *Institut fur Biophysik und Strahlenbiologie der Universitat Freiburg, Albertstrasse 23, D-7800 Freiburg, Federal Republic of Germany; and tDepartment of Ophthalmology and tDepartment of Biochemistry and Molecular Biology, University of Florida, Gainesville, FL 32610 Communicated by L. M. Beidler, June 8, 1989 (receivedfor review February 8, 1989) ABSTRACT Rhodopsin is a member of an ancient class of binding of Gt to photolyzed rhodopsin therefore abolish the receptors that transduce signals through their interaction with extra MII. The extent of reduction of the extra MII by a guanine nucleotide-binding proteins (G proteins). We have competitor measures the effectiveness of the competitor in mapped the sites of interaction ofrhodopsin with its G protein, disrupting the MII-Gt interaction. This assay represents a which by analogy suggests how other members of this class of method of high sensitivity and high specificity to probe the receptors may interact with their G proteins. Three regions of nature of the interaction between photolyzed rhodopsin and rhodopsin's cytoplasmic surface interact with the rod cell G Gt. We previously showed (9) that selected peptides from the protein transducin (Ga. These are (i) the second cytoplasmic sequence of the Gt a subunit (Gta) can interfere with binding loop, which connects rhodopsin helices m and IV, (U) the third of Gt to photolyzed rhodopsin, thus allowing assignment of cytoplasmic loop, which connects rhodopsin helices V and VI, these peptides to the region ofthe Gta sequence that binds to and (iu) a putative fourth cytoplasmic loop formed by amino rhodopsin. In the work described here, we tested peptides acids 310-321, as the carboxyl-terminal sequence emerges from from the rhodopsin sequence in order to see which ones helix VII and anchors to the lipid bilayer via palmitoylcysteines reduce the level of extra MII. Such peptides presumably 322 and 323. Evidence for these regions of interaction of would do so by simulating a region ofrhodopsin's surface that rhodopsin and Gt comes from the ability of synthetic peptides interacts with Gt, thus interfering with Gt binding to MII. comprising these regions to compete with metarhodopsin II for binding to Gt. A spectroscopic assay that measures the "extra MATERIALS AND METHODS MIU" caused by Gt binding was used to measure the extent of binding of Gt in the presence of competing peptides. The three Spectrophotometric Assay. Binding of Gt to MII was mea- peptides corresponding to the second, third, and fourth cyto- sured as in refs. 7-9. The assay was performed at pH 8 and plasmic loops competed effectively with metarhodopsin H, 4°C, conditions under which only a small, control amount of exhibiting Kd values in the 2 jtM range; 11 additional peptides MII is formed in the absence of Gt. The full extra MII signal comprising all remaining surface regions of rhodopsin failed to in the presence ofGt corresponds to a 60% MII fraction ofthe compete even at 200 ,M. Any two peptides that were effective total photoexcited rhodopsin. The final levels of MII forma- competitors showed a synergistic effect, having 15 times higher tion minus the control level (no Gt present) are a direct effectiveness when mixed than when assayed separately. A measure of the rhodopsin-Gt complexes formed. When nor- mathematical model was developed to describe this behavior. malized to the undisturbed full extra MII signal, they yield the relative amount of Gt that is able to interact with rhodopsin. Rhodopsin is the best-studied receptor protein ofthat class of Washed disk membranes were prepared from bovine rod signal-transducing receptors that act via guanine nucleotide- photoreceptors as described (8) and were suspended in buffer binding proteins, or G proteins. Other members of this class A (100 mM NaCl/2 mM MgCl2/2 mM CaC12/0.2 mM EDTA/ include the adrenergic receptors (1), the muscarinic acetyl- 1 mM dithiothreitol/40 mM Hepes, pH 8.0) in a cuvette at choline receptors (2), the substance K receptor (3), and 4°C; all measurements were made at a final rhodopsin con- dozens of other receptors including those for neurotransmit- centration of 1.5 ,M. Purified (10) Gt (0.75 AM final concen- ters, peptide hormones, and other regulatory factors (4). In tration) and peptides (see below), also in buffer A, were the retinal rod cell rhodopsin is excited by light and under- added to the disk membranes. Most measurements were goes a change in conformation that allows it to activate the G performed with purified Gt; use of low-ionic-strength extract protein transducin (Gt), which in turn activates a cGMP gave identical results. In one case (peptide CIV), dithiothrei- phosphodiesterase (reviewed by Stryer, ref. 5). In this report tol was replaced by 2 mM ascorbate. Control experiments we identify those sites on the surface of photoexcited with other peptides using ascorbate instead of dithiothreitol rhodopsin that are involved in the interaction with Gt. Similar gave the same results. Peptide LII was dissolved in a mini- surface sites on homologous receptors may serve to bind and mum quantity of ethanol because of its limited solubility in to activate their G proteins. buffer. When rhodopsin is stimulated by light it relaxes within Peptide Synthesis. Peptides were synthesized by the solid- milliseconds to an equilibrium between two tautomeric phase Merrifield method with an Applied Protein Technolo- forms, metarhodopsin I and II (MI and MII) (reviewed in ref. gies synthesizer (Cambridge, MA) and were purified by 6). MII interacts strongly with Gt, shifting the equilibrium to high-performance liquid chromatography (11). Peptides from form "extra MII" (6-8). The extra amount of MII is a rhodopsin's cytoplasmic (C) surface or lumenal (L) surface in stoichiometric measure ofthe MII-Gt complexes formed and the disk membrane (see Fig. 4) had the following amino acid can be easily measured spectroscopically following a flash of sequences: CI, residues 61-75; CII, 141-153; CIII, 230-252; light due to the large difference between the absorption CIV, 310-321; CV, 323-334; CVI, 327-338; CVII, 337-348; maxima of MI and MII (8). Substances that compete for LI, 13-23; LII, 96-115; LIII, 188-203; LIV, 276-286. An analog of peptide CII was made in which the amino acids The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" Abbreviations: G protein, guanine nucleotide-binding protein; Gt, in accordance with 18 U.S.C. §1734 solely to indicate this fact. transducin; MI and MII, metarhodopsins I and II. 6878 Biochemistry: K6nig et al. Proc. Natl. Acad. Sci. USA 86 (1989) 6879 141-152 were "scrambled," or assembled randomly (se- rhodopsin peptides; e.g., when peptide CI, which connects quence Phe-Ser-His-Gly-Arg-Met-Asn-Pro-Glu-Lys-Asn- rhodopsin helices I and II, was added to the rhodopsin/Gt Phe). mixture, the amount of extra MII formed on photolysis was not affected. Note that the signals obtained for the high concentration of effective peptide are kinetically identical to RESULTS those of the control. This indicates that in the presence of Measurements of Extra MII Show How Much Gt Is Bound peptide, the photochemical reaction proceeds normally to the to MIR. When rhodopsin is photolyzed, an amount of MII is MI/MII equilibrium. We conclude that the peptide prevents formed that is characteristic of the particular reaction con- interaction of Gt with MIT but does not affect formation of ditions (Fig. 1A, trace i). Additional MII is formed when Gt MIT itself. is present, due to its binding to MIT and shifting the MI/MII Peptides from Three Regions of Rhodopsin's Cytoplasmic equilibrium (Fig. lA, trace ii). We found that certain peptides Surface Are Effective Competitors. Fig. 2 shows the depen- from the rhodopsin sequence were able to inhibit this effect dence ofextra MII formation on peptide concentration for 11 of Gt and reduce the amount ofextra MIT. Rhodopsin peptide peptides that comprise the aqueous-exposed surface of CIV, which comprises rhodopsin's cytoplasmic sequence rhodopsin. Three of the peptides (CII, CIII, and CIV) were 310-321, reduced the amount ofextra MII when present at 15 effective in preventing G, binding to rhodopsin (Fig. 2). These and 75 ILM (Fig. 1B, traces i and ii). When present at 300 ILM, peptides are from the cytoplasmic surface ofrhodopsin. They peptide CIV completely abolished the extra MII so that the form loops connecting helices III and IV (CII), helices V and amount of MII formed was the same as in the absence of Gt VI (CIII), and helix VII and its site of anchoring in the disk (Fig. 1B, trace iii). This effect is specific for only certain membrane (CIV, palmitic acids esterified to Cys-322 and Cys-323; ref. 13) (see Fig. 4). By contrast, peptides repre- senting other regions of the cytoplasmic surface were inef- A Mll fective in this assay: e.g., the loop connecting helices I and II (CT) and the carboxyl-terminal region 323-348 (CV, CVI, and CVII; Fig. 2). As expected, none of the peptides repre- senting regions of rhodopsin's surface exposed to the disk lumen were able to perturb the binding of Gt to MII (Fig. 2). Extra MlI Peptide CII with a "scrambled" sequence was completely (ii) ineffective; the same was found for a peptide comprising only the amino-terminal part (residues 231-241) of the CIII se- quence (data not shown).