Transducin-alpha C-terminal Peptide Binding Site Consists of C-D and E-F Loops of Rhodopsin*

(Received for publication, September 27, 1996, and in revised form, December 3, 1996)

Shreeta Acharya , Yasser Saad and Sadashiva S. Karnik Dagger

From the Department of Molecular Cardiology, Research Institute, The Cleveland Clinic Foundation, Cleveland, Ohio 44195-5069

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
Acknowledgments
Note Added in Proof
REFERENCES


ABSTRACT

The binding of heterotrimeric GTP-binding proteins (G-proteins) to serpentine receptors involves several independent contacts. We have deduced the points of interaction between mutant bovine rhodopsins and alpha t-(340-350), a peptide corresponding to the C terminus of the alpha  subunit (alpha t) of bovine retinal G-protein, transducin. Direct binding of alpha t-(340-350) to rhodopsin stabilizes the activated metarhodopsin II state (M II), consequently uncoupling the rhodopsin-transducin interaction. This peptide action requires two segments on the cytoplasmic domain of rhodopsin: the Tyr136-Val137-Val138-Val139 sequence on the C-D loop and the Glu247-Lys248-Glu249-Val250-Thr251 sequence on the E-F loop. We propose that a tertiary interaction of these two loop regions forms a pocket for binding the alpha t C terminus of the transducin during light transduction in vivo. In most G-proteins, the C termini of alpha  subunits are important for interaction with receptors, and, in several serpentine receptors, regions similar to those in rhodopsin are essential for G-protein activation, indicating that the interaction described here may be a generally applicable mode of G-protein binding in signal transduction.


INTRODUCTION

Activation of heterotrimeric guanine nucleotide-binding proteins (G-proteins)1 by transmembrane receptors is a general paradigm for signal transduction by a large variety of hormones, neurotransmitters, and physical stimuli. The G-protein coupled receptors (GPCRs) contain an extracellular N-terminal tail, seven transmembrane helices, three interhelical loops on either side of the membrane, and a cytoplasmic C-terminal tail. The cytoplasmic domain of the receptors binds and activates the G-protein (1-5). Visual transduction in rod cells is a prototypical example of a G-protein-coupled signaling system. In rod cells, signal transduction is initiated by photon-induced isomerization of the 11-cis-retinal chromophore, to all-trans-retinal. As shown in Fig. 1, this generates an inactive intermediate, metarhodopsin I (M I), and structural changes in the apoprotein leads to an active intermediate, metarhodopsin II (M II). The M II then binds and activates the retinal G-protein, transducin (Gt). Evidence from peptide competition (6), mutational (7-9), and biochemical (10) studies have implicated three cytoplasmic regions of M II as being critical for Gt interaction. Likewise, in transducin, the alpha  subunit residues 340-350 at the C terminus, 311-323 at alpha 4/beta 6/alpha 5 regions, 8-23 at the N terminus, and the farnesylated at the C-terminal tail of the gamma 1 subunit have been shown to be specific contact sites for rhodopsin (11, 12). Additional contact sites involving the beta  subunit are anticipated but have not been mapped. Thus, several distinct contacts are involved in the signal transfer from rhodopsin to Gt, but which segment of Gt interacts specifically with a particular region of rhodopsin is not known.


Fig. 1. Schematic representation of the steps involved in the stabilization of the M II state, GDP-GTP exchange, and the alpha t-(340-350)-mediated inhibition of Gt activation.
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This paper focuses on the identification of the residues of bovine rhodopsin that interact with the transducin alpha  subunit C-terminal residues 340IKENLKDCGLF350, a region that is important in rhodopsin-transducin coupling (11, 13-16). The ability of an 11-amino acid alpha t-(340-350) peptide to directly stabilize the M II state of rhodopsin mutants was employed. We report that the binding site consists of the residues Tyr136 through Val139 in the C-D loop and the residues Glu247 through Thr251 in the C-terminal portion of the E-F loop of bovine rhodopsin.


EXPERIMENTAL PROCEDURES

Expression, Purification, and Characterization of Mutant Rhodopsins

Procedures for the construction of mutants and expression of opsins have been described earlier (17, 18). Wild-type and mutant opsin genes (Table I) were expressed in COS1 cells by transient transfection of corresponding gene. The rhodopsin chromophore was generated by adding 11-cis-retinal (40 µM) to a cell suspension, the cells were solubilized in 1% dodecyl maltoside, and rhodopsin was purified by immunoaffinity chromatography (17, 18). The pigment concentration was calculated from its absorbance at 500 nm based on E500 = 42,700 M-1 cm-1. For rhodopsin samples prepared for M I left-right-arrow  M M II equilibrium studies, the dodecyl maltoside was replaced with 1% digitonin in all washes and elution.

Table I.

Influence of rhodopsin cytoplasmic domain mutants

Transducin activation by mutant and wild-type rhodopsin were carried out as described under "Experimental Procedures." The relative mean GTPgamma S binding values (S.E. < 5%) are equilibrium values obtained without any correction for the decay of metarhodopsin II that might have occurred during the 60-min assay period. The results presented are a mean of 3 to 5 separate experiments in which mutant and wild-type rhodopsin expressed in COS cells were assayed in parallel and the mutant activities were normalized to the wild-type activity. The concentration of opsin was estimated from the lambda max assuming a molar extinction coefficient of 42,000 mol-1 cm-1.
Mutant Sequencea Transducin activation (fraction of wild-type) Peptide 1 bindingb

C-D loop
134               150
  Wild-type   ERYVVVCKPMSNFRFGE   1.00 +
  CD1   ---AAAA---------- 0.69 +/-
  CD2   --LAAAA---------- 0.22  -
  CD3   -------EPNHMFN--- 0.55 +
  CD4   ---------NHMFN--- 0.45 +
  CD5   -------EPNHM----- 0.44 +
E-F loop
231                    252
  Wild-type   KEAAAQQQESATTQKAEKEVTR 1.00 +
  EF1   ----TSLHGYSVTGPTGSNL-- 0.09  -
  EF2   ----TSLHGYSV---------- 0.59 +
  EF3   -----------GG--------- 0.45 +
  EF4   -------------GLTGSNL-- 0.09  -
  EF5   -------------GLT---L-- 0.29 +
  EF6   -------------------GG- 0.58 +/-

a Wild-type bovine rhodopsin amino acid sequence corresponds to arbitrarily assigned interhelical regions. The solid lines indicate the amino acid sequence not altered in the specific mutant.
b Significant shift of M II/M I equilibrium toward formation of M II in the presence of 500 µM alpha 1-(340-350) peptide 1.

Transducin Activation

Transducin was isolated from the bovine rod outer segment as described by Fung et al. (19). Catalytic activation of transducin by wild-type and mutant rhodopsins was assayed by a (GTPgamma S) binding assay as described by Wessling-Resnick and Johnson (20). The assay mixtures consisted of 1-5 nM purified rhodopsin, 2 µM transducin, 20 µM [35S]GTPgamma S (1130 Ci/mmol) in 10 mM Tris-HCl, pH 7.2, 100 mM NaCl, 5 mM dithiothreitol, and 0.012% dodecyl maltoside. The assay was initiated by illumination for 2 min at a wavelength greater than 495 nm. The reaction mixture then remained in the dark at 23 °C for 60 min. The number of moles of [35S]GTPgamma S bound per mol of rhodopsin in 60 min was estimated from the [35S]GTPgamma S retained on the filter after filteration and washing.

Synthesis and Characterization of Peptides

The alpha t-(340-350) peptide, Ac-IKENLKDCGLF, and seven analogues (Fig. 2A) were synthesized, purified, and characterized by the protein chemistry core services of the Research Institute of the Cleveland Clinic as described earlier (18). These peptides will be referred to as peptides 1 (the parent peptide) through 8. 


Fig. 2. Effect of alpha t-(340-350) analogues on the [35S]GTPgamma S binding to Gt stimulated by light-activated rhodopsin (A) and the formation of M II state from bleached rhodopsin (B). The [35S]GTPgamma S binding assay mixtures consisted of 1 nM purified rhodopsin, 2 µM transducin, 20 µM [35S]GTPgamma S, and 0.012% dodecyl maltoside. The bars represent maximal binding in the presence of 500 µM alpha t-(340-350) analogues at 23 °C for 60 min. In separate experiments 1-1000 µM concentrations of each peptide analogue were used to obtain inhibition curves, and apparent the Ki values shown were estimated by linear regression analysis of the inhibition curves. The values represent the mean of three measurements on the same batch of purified rhodopsin. The Ki values have a congruent 15% error. The M II stabilization assay used rhodopsin samples in 1% digitonin at rhodopsin:alpha t-(340-350) ratios of 1:700. Representative spectra yielded by four alpha t-(340-350) analogues with a significant affinity change are shown. Please refer to Fig. 3 for the wild-type spectrum with peptide 1. The samples in each case were: dark rhodopsin + 500 µM peptide (1), light-activated rhodopsin without the peptide (2), and light-activated rhodopsin + 500 µM peptide (3).
[View Larger Version of this Image (31K GIF file)]


The alpha t-(340-350)-induced M I left-right-arrow  M II Equilibrium Assay

The principles and procedure for the M I left-right-arrow  M II equilibrium assay have been described previously (16, 18). In a typical experiment, ~5 to 8 × 10-8 M rhodopsin was evaluated with 1 × 10-4, 5 × 10-4, and 1 × 10-3 M concentrations of each peptide. The alpha t-(340-350) peptide or its analogues were mixed with wild-type and mutant rhodopsins in 1% digitonin in the dark and kept on ice for 20 min. Dark spectra were recorded at 5 °C. The samples were then exposed to light for 20 s using a 150-watt Fiber-Lite fitted with 490 nm cut-off filter; the sample was allowed to equilibrate in the dark at 5 °C for 20 min, and then light spectra were recorded (see Figs. 2B and 3).


Fig. 3. Effect of alpha t-(340-350) on M I left-right-arrow  M II equilibrium of rhodopsin mutants in 1% digitonin. 1, dark rhodopsin + 500 µM peptide 1; 2, light-activated rhodopsin without peptide 1; 3, light-activated rhodopsin + 500 µM peptide 1. The molar ratio of wild-type rhodopsin to peptide 1 was ~1:1400, and the ratio of various mutant rhodopsins to peptide 1 varied from 1:3000 to 1:13,000.
[View Larger Version of this Image (17K GIF file)]



RESULTS AND DISCUSSION

The conclusions in this study are based on the analysis of the ability of the alpha t-(340-350) peptide to directly bind and stabilize the M II intermediate. Two different assays were employed: (i) alpha t-(340-350) inhibition of rhodopsin-stimulated transducin activation and (ii) formation of M II from the M I intermediate by alpha t-(340-350)-dependent stabilization of M II. The relationship between these two assays is schematically shown in Fig. 1. Previous studies have found that bleaching rhodopsin in dodecyl maltoside in the absence of Gt yields the active M II state with a lambda max ~380 nm, an intermediate that stimulates [35S]GTPgamma S binding to Gt (steps 2 through 7). In contrast, bleaching rhodopsin in 1% digitonin in the absence of Gt yields predominantly the inactive M I (lambda max ~480 nm) intermediate (step 2) and a small amount of M II (7, 17, 18, 21). Formation of the M II state in digitonin requires a stoichiometric mixture of rhodopsin and Gt because instantaneous stabilization of M II by Gt is necessary (steps 3 and 5). Adding the non-hydrolyzable GTP analogue, GTPgamma S, destabilizes the M II:Gt complex (step 7) and shifts the equilibrium in favor of the M I state (21). The formation of the M II state in digitonin can also be induced by alpha t-(340-350) rather than Gt (steps 3 and 4). The stabilization of the M II state depends on the amino acid sequence and concentration of the alpha t-(340-350) peptide (11, 18). Thus, alpha t-(340-350), through binding to a site on rhodopsin, competes with Gt to shift the M I left-right-arrow  M II equilibrium and thus inhibits [35S]GTPgamma S binding to Gt (step 6).

To identify the site on rhodopsin required for binding alpha t-(340-350), wild-type and mutant rhodopsins purified from transfected COS1 cells were used in both assays. The mutant opsins (Table I) expressed well (expression was estimated by Western blot analysis, not shown), bound 11-cis-retinal and yielded a chromophore within 90% of that yielded by the wild-type, indicating that the mutant polypeptides folded normally to a native state. All mutant rhodopsins purified in dodecyl maltoside and digitonin yielded a chromophore with a lambda max ~500 nm (data not shown). Light-activated rhodopsin samples in dodecyl maltoside were used for measuring [35S]GTPgamma S binding to Gt (Fig. 2A and Table I). The wild-type rhodopsin activated nearly 267 ± 29 mol of Gt/mol of rhodopsin. Unregenerated opsin and rhodopsin not exposed to light activated 15 ± 2 mol of Gt/mol of rhodopsin.

The synthetic 11-amino acid alpha t-(340-350) peptide 1 inhibited (50 ± 10% of maximal) Gt activation by bleached rhodopsin. The apparent Ki for inhibition by peptide 1 was 80 µM. In agreement with earlier studies using urea-washed rod outer segment disc membranes, an inhibition greater than 50% could not be achieved at a higher peptide 1 concentration (11). As shown in Figs. 2B and 3, bleaching wild-type rhodopsin in 1% digitonin yielded predominantly the M I intermediate (lambda max ~480 nm) and a small amount of the M II intermediate. The peptide did not affect the spectrum of rhodopsin in the dark. The presence of 500 µM alpha t-(340-350) yielded approx 50% M II at the expense of M I. Comparing the potency of the peptide in the two assays indicated that an approximately 200-fold molar excess of peptide 1 was required for half-maximal inhibition of [35S]GTPgamma S binding to Gt (Ki = 80 µM), and an approximately 1000-fold molar excess of peptide was needed for a half-maximal shift of M I to M II. Thus, the interaction of alpha t-(340-350) and Gt with the detergent-solubilized wild-type rhodopsin obtained from COS1 cells had properties identical to those reported for interaction with bovine retinal rhodopsin (11).

Nature of the Interaction between alpha t-(340-350) and M II

To determine which amino acid side chains of alpha t-(340-350) are important for M II stabilization, the synthetic analogues shown in Fig. 2A were used. The C-terminal 347CGLF350 sequence was not examined because this region has already been shown to form a beta -turn structure, and the subtype of the beta -turn is speculated to be important for receptor selectivity (16, 22). Conservative single amino acid substitution of the remaining seven residues led to varying phenotypes (Fig. 2). Peptides 2 and 8 competed as effectively against Gt as the parent peptide 1 in both assays. Peptide 5 was a slightly (approx 2-fold) more effective competitor of Gt and also better shifted the M I left-right-arrow  M II equilibrium in favor of M II (Fig. 2, A and B). Peptide 3 had a slightly lower potency (approx 2.5-fold less) than peptide 1. Peptides 4, 6, and 7 were very poor competitors in the Gt activation assay and were completely ineffective in shifting the M I left-right-arrow  M II equilibrium (Fig. 2B).

Studies using transferred nuclear Overhauser effect spectroscopy suggested that a salt bridge between Glu342 and Lys345 exists in alpha t-(340-350) bound to rhodopsin in the dark, that is broken during M II stabilization and replaced by a new salt bridge between Lys345 and the free alpha -COO- group of the peptide (16). The Gln substitution can provide hydrogen bonding interactions in the place of a salt bridge. However, the Lys345 right-arrow Gln change (peptide 7) is likely to affect both conformations required for binding to rhodopsin, in the dark, as well as M II. But, the Glu342 right-arrow Gln change in peptide 4 is expected to favor the conformation that enables M II stabilization. The lack of peptide 4 binding suggests that Glu342 is essential for stabilizing M II state. Lys341 made a negligible contribution. Leu344 is a critical residue. Substitution with shorter Ala (peptide 6) produced an inactive peptide indicating that hydrophobicity and the side chain size of Leu344 side chain are stringent requirements for interaction with rhodopsin. Consistent with this observation, Martin et al. (23) discovered that combinatorial analogues of alpha t-(340-350) preserve the shape of the hydrophobic "face" with little variation, whereas larger changes in the hydrophilic face are tolerated. Mutagenesis studies suggest a binding preference for hydrophobic amino acids at the Leu344 and Leu349 positions of alpha t (13, 14). Thus, the site on rhodopsin that binds alpha t-(340-350) is expected to consist of charged and hydrophobic residues. Furthermore, the analogue and mutagenesis studies combined together indicate that the alpha t-(340-350) peptide and residues 340-350 of Gt are similar in their interactions with M II and therefore very likely bind to a common site on rhodopsin.

Localization of alpha t-(340-350) Binding Residues of Rhodopsin

To identify the rhodopsin alpha t-(340-350) binding site, we created rhodopsin mutants in three distinct cytoplasmic regions involved in the Gt interaction. Amidst these, it should be possible to identify mutants in which the alpha t-(340-350) binding is abolished even though the interaction with Gt is not completely abolished. The abolished alpha t-(340-350) binding should be restored upon re-introduction of the wild-type amino acid sequence. Five C-D loop mutants and six E-F loop mutants were chosen for analysis. In these mutants, formation of rhodopsin and M II-like states determined by spectral analysis was not altered, but the activation of Gt by the mutant M II was altered to various degrees (Table I). The mutants of the remaining cytoplasmic region implicated in Gt binding (residues 310-322 at the membrane-proximal carboxyl tail region of rhodopsin) had a low yield of a rhodopsin-like chromophore and M II-like photo product. Hence, they were not examined further.

In a previous study we concluded that the residues Glu134 and Arg135 in the C-D loop are not essential for alpha t-(340-350) binding (18). When the highly conserved Tyr136 residue was substituted with Gly, the resulting mutant bound retinal poorly (data not shown). The mutant CD1, in which the residues 137-140 were replaced by four alanines to preserve the alpha -helical potential but alter the amino acid sequence, transducin activation was reduced approx 30%. As shown in Fig. 3, the M II yield was approx 20% in the presence of approx 2000-fold excess of alpha t-(340-350). To specifically determine the role of the Tyr136 residue in the context of the CD1 mutant sequence, the residues 136-140 (YVVVC of the wild-type) were replaced with LAAAA (mutant CD2). Transducin activation was reduced approx 80%. A nearly 6000-fold excess of alpha t-(340-350) did not shift the M II left-right-arrow  M I equilibrium (Fig. 3). Thus, Tyr136 is an important determinant for alpha t-(340-350) stabilization of the M II state. Previously, Ridge et al. (24) used cysteine scanning mutagenesis to demonstrate that individual replacement of Tyr136, Val137, Val138, and Val139 led to partial loss of Gt activation. The Cys140 residue was found to be not essential (17, 24). Therefore, the VVV sequence following Tyr136 may contribute stabilizing interactions. In our study, the remaining substitution mutants (CD3, CD4, and CD5) caused partial loss of Gt activation but showed normal affinity for the alpha t-(340-350) (data not shown). Thus, the residues 141-150 of the C-D loop do not participate in alpha t-(340-350) binding.

Franke et al. (7) found that replacing the E-F loop region between residues 231 and 252 with an amino acid sequence from the extracellular loop B-C produced a rhodopsin molecule that was normally activated by light but stimulated transducin very poorly. We constructed the same mutant (EF1 in Table I). The mutant exhibited normal photocycle properties as reported earlier and also activated transducin at only approx 9% of the wild-type control. This mutant produced an M I-like state when bleached in digitonin. The M II left-right-arrow  M I equilibrium of the mutant was not shifted by alpha t-(340-350), suggesting that this mutant lacks the binding site for the peptide (Table I and Fig. 3).

We constructed mutants EF2 through EF5 by restoring the wild-type amino acid sequence in different parts of the E-F loop region. The mutant EF6 was constructed to examine the remaining two residues predicted to be the part of the E-F loop. As indicated in Fig. 3 and Table I, mutants EF2, EF3, and EF6 activated transducin at approx 40-60% of the wild-type. The mutants EF2 and EF3 bound alpha t-(340-350) almost as well as the wild-type. The mutant EF6 exhibited an interesting phenotype. The alpha t-(340-350) binding was evident because the M I peak decreased. However, this decrease was not accompanied by a transition to a distinct M II peak but rather by an increase in light scattering at the spectral region below 380 nm. The EF6 mutation must either alter the affinity for alpha t-(340-350) or decrease the stability of the M II·alpha t-(340-350) complex. Therefore, removing the Val250-Thr251 side chains likely indirectly influences the alpha t-(340-350) interaction. The mutant EF4 was essentially inactive in both the peptide binding and Gt stimulation assays. Examination of the residues in this mutant indicates that hydrophilic and charged residues present in the wild-type rhodopsin E-F loop are replaced by hydrophobic (Leu), shorter (2 Gly residues), and hydrogen-bonding (Asn, Ser, and Thr) residues.

On the basis of earlier mutagenesis studies, the Glu247-Lys-Glu-Val-Thr251 sequence is thought to be essential for efficient activation of transducin and that the other residues play a relatively minor role (7, 9). The EKE triad sequence was kept in the mutant EF5. The M II left-right-arrow  M I mixture generated by bleaching this mutant was shifted toward M II formation by alpha t-(340-350), suggesting that the peptide was now able to bind and stabilize the M II intermediate. Transducin activation was partially restored (approx 30%). Therefore, it seems reasonable that the charged triad Glu247-Lys248-Glu249 is necessary for the M II left-right-arrow  M I equilibrium shift promoted by alpha t-(340-350) binding. We conclude that alpha t-(340-350)-mediated stabilization requires the hydrophobic residues Tyr136-Val-Val-Val139 on the C-D loop and the hydrophilic charged residues Glu247-Lys-Glu-Val-Thr251 on the E-F loop of rhodopsin. The type of analysis used here is not sensitive enough to determine which side chains of the alpha t-(340-350) interact with each of the regions.

Fig. 4 depicts the location of the two sites required for alpha t-(340-350) binding in the cytoplasmic extensions of the C and F helices. In conventional models, the transmembrane helices of rhodopsin are terminated at the membrane-aqueous interface. However, recent site-directed spin-labeling studies suggest that approx 1 to 3 turns of the helices extend into the cytoplasm, with helix C having a close tertiary interaction with helices B, D, E, and F. In a revised model, the Tyr136 and Val139 of helix C faces helix F, and Lys247 of helix F faces helix C (24, 25). This observation supports our hypothesis that the tertiary interaction of Tyr136-Cys140 and Glu247-Thr251 regions forms a subsite that is stabilized by alpha t-(340-350). The spin-labeling studies suggest that both these segments are rigid relative to the helix E extension, which is more dynamic and not essential for binding the alpha t-(340-350). Based on these observations, some qualitative conclusions can be drawn regarding M II stabilization by alpha t-(340-350) and holotransducin in vivo. Perhaps the rigidity of the cytoplasmic helix C and helix F extensions is required to provide an optimal surface for binding. The M II stabilization may occur because entropy is lost after alpha t-(340-350) has bound to the rigid cytoplasmic extensions of helices C and F. This loss explains M II·Gt complex stabilization by the Gt-alpha residues 340-350, which are currently believed to be disordered in the heterotrimer (26). It is noteworthy that these two rhodopsin helices contact the ionone ring of the 11-cis-retinal chromophore (27, 28).


Fig. 4. A model of the cytoplasmic C-D and E-F loops of rhodopsin adopted from Ref. 25. The residues forming the binding site for alpha t-(340-350) are highlighted.
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It is now generally assumed that the G-protein binding site of all GPCRs comprises regions from the C-D loop, E-F loop, and the membrane-proximal segment of the cytoplasmic tail (1-9). Various types of studies indicate that the E-F loop is preeminent in the G-protein activation process in GPCRs (3-5). A hydrophobic site near the N-terminal region of the E-F loop that is important for G-protein coupling in several GPCRs (4, 29) appears not to be crucial for alpha t-(340-350) interaction with rhodopsin. Instead, our results indicate that the alpha t-(340-350) binding involves a hydrophobic region of the C-D loop. The hydrophilic and charged portion of this pocket near the C terminus of the E-F loop corresponds to a site that is important for G-protein activation in several GPCRs (4). E-F loop regions of different GPCRs were found to cross-link to specific alpha -subunits (30), as well as to beta -subunits of G-protein heterotrimers (31). All these evidences suggest that the E-F loop may wrap around the G-protein heterotrimer, establishing contacts with critical regions of the alpha -subunit, as well as with the beta gamma complex. Our results are the first description of an interaction between a defined region on transducin and a specific site on the receptor. Our approach could be used to explore the three other subsites on rhodopsin for Gt regions, alpha t-(311-323), alpha t-(8-23), and the farnesylated gamma t-(60-71) residues.


FOOTNOTES

*   This work was supported in part by NEI Grant 09704 from the National Institutes of Health.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Dagger    To whom correspondence should be addressed: Dept. of Molecular Cardiology, FF3-27, Research Institute, Cleveland Clinic Foundation, 9500 Euclid Ave., Cleveland, Ohio 44195. Tel.: 216-444-1269; Fax: 216-444-9263.
1   The abbreviations used are: G-proteins, guanine nucleotide-binding proteins; GTPgamma S, guanosine 5'-O-(3-thiotriphosphate); M I, metarhodopsin I; M II, metarhodopsin II; Gt, transducin heterotrimer; alpha t or Gt-alpha the alpha  subunit of transducin; Gt-beta gamma , the beta gamma subunit complex of transducin; alpha t-(340-350), synthetic peptide corresponding to the C-terminal 11-amino acid sequence of the alpha  subunit of transducin; GPCR, G-protein-coupled receptor.

Acknowledgments

We are indebted to Dr. Kunio Misono for assistance in synthesis and characterization of peptides; Dr. R. Crouch, Medical School of South Carolina, Charlotte, SC for supplying 11-cis-retinal, Dr. Ramaswamy Ramachandran for critical reading of the manuscript, and Robin Lewis and Cassandra Talerico for manuscript preparation.


Note Added in Proof

During the review of this manuscript two papers were published (Farrens, D. L., Altenbach, C., Yang, K., Hubbell, W. L., and Khorana, H. G. (1996) Science 274, 768-770; Sheikh, S. P., Zvyaga, T. A., Lichtarge, O., Sakmar, T. P., and Bourne, H. R. (1996) Nature 383, 347-350). Using two different experimental systems the authors have reached the same conclusion which indicates that movement of transmembrane helices C and F is required for light activation of rhodopsin. Furthermore these studies demonstrated the proximity of cytoplasmic extensions of transmembrane helices C and F which are identical to the segments identified in our study.


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