Receptor activation: what does the rhodopsin structure tell us?

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Receptor activation: what does the rhodopsin structure tell us? Elaine C. Meng and Henry R. Bourne G-protein-coupled receptors (GPCRs) are a large family of seventransmembrane-helix proteins that mediate responses to hormones, neurotransmitters and, in the case of rhodopsin, photons. The recent determination of the structure of rhodopsin at atomic resolution opens avenues to a deeper understanding of GPCR activation and transmembrane signaling. Data from previous crosslinking, spin labeling and scanning accessibility experiments on rhodopsin have been mapped onto the highresolution structure. These data correlate well and are consistent with the structure, and suggest that activation by light opens a cleft at the cytoplasmic end of the seven-helix bundle of rhodopsin. Furthermore, lessons learned from rhodopsin might also apply to other members of this essential family of receptors. (For an animation of the crystal structure of rhodopsin see http://archive.bmn.com/supp/tips/tips2211a.html)

Elaine C. Meng Depts of Cellular and Molecular Pharmacology and Pharmaceutical Chemistry Henry R. Bourne* Depts of Cellular and Molecular Pharmacology and Medicine, University of California, San Francisco, CA 94143-0450, USA. *e-mail: [email protected]

The first high-resolution structure of a G-proteincoupled receptor (GPCR), rhodopsin1, generates many questions. What does the structure tell us that is new? How accurate were our inferences about the structure, based on previous experiments in many laboratories? Does the structure allow us to generalize from rhodopsin to other GPCRs? Most importantly, can the structure explain how light activates rhodopsin? This review will try to answer these questions. Many previous inferences were correct, but the structure also tells us much that is new. Furthermore, the structure of rhodopsin provides an excellent model for other GPCRs. Although the new structure shows the unactivated, ‘dark state’ rhodopsin, it confirms a crude but consistent picture of lightinduced movements in the receptor’s bundle of transmembrane helices. In this review, data from previous crosslinking, site-directed spin labeling, and scanning accessibility determinations on rhodopsin will be summarized. Together, the data and the structure suggest that activation by light opens a cleft at the cytoplasmic end of the helix bundle, with separation of transmembrane helices III and VI and increased exposure of the inner faces of II, III, VI and VII. Decreases in exposure occur near the ends of IV and V. The implications of the new structure for understanding GPCRs and additional avenues for possible experiment will also be discussed. Rhodopsin structure

Unlike most GPCRs, rhodopsin binds its ligand, 11-cis-retinal, covalently. An encounter with a photon isomerizes the ligand into the all-trans conformation, which acts as a covalently bound agonist, triggering http://tips.trends.com

receptor activation by altering the conformation of rhodopsin. As with other GPCRs, activated rhodopsin catalyzes replacement by GTP of GDP bound to the α-subunit of a heterotrimeric G protein, in this case transducin, which interacts with cytoplasmic loops of the receptor. In turn, dissociation of the GTP-bound α-subunit (αt–GTP) from the βγ heterodimer enables αt–GTP to trigger the rod cell’s response to light. In the high-resolution structure of rhodopsin1 (Fig. 1a), 11-cis-retinal is located far from the cytoplasm, in a pocket formed by the transmembrane helices near the extracellular (EC) side of the membrane. Isomerization of 11-cis-retinal to all-trans-retinal must nudge transmembrane helices into a new ‘on’ conformation that is different from the ‘off ’ state observed in the crystal structure. Now that the ‘off ’ state conformation is known with much greater precision, we should be able to generate more precise hypotheses about how the receptor transmits the photon message from the binding pocket to the cytoplasmic loops, which contact the G protein. Previous low-resolution electron density maps had roughly outlined the positions of the transmembrane helices2,3, and the high-resolution structure (Fig. 1a) is consistent with these maps. In addition to the seven-helix transmembrane bundle, the new structure shows, for the first time, the N-terminus and all three EC loops, plus (with two short gaps) the three intracellular (IC) loops and the C-terminal tail. The EC and IC structures offered several surprises, including a compact EC ‘lid’, parts of which fold inwards to completely enclose retinal, and an eighth helix (VIII) on the IC side, approximately parallel to the plane of the membrane and perpendicular to the seven-helix bundle. The structure of rhodopsin’s transmembrane helix bundle was predicted, with considerable precision, in previous models. All the models4–6 that are now evident as the most successful (3.1–3.2 Å root mean square deviation for ∼200 α-carbons in common with the crystal structure) were guided by the lowresolution electron density of rhodopsin2,3,7 and by patterns of conservation in the amino acid sequences of other GPCRs. This level of accuracy suggests that the transmembrane bundle structure is indeed conserved among GPCRs, and possibly within the entire GPCR family. By contrast, the loops and termini are more divergent in amino acid sequence and probably in three-dimensional (3D) structure.

0165-6147/01/$ – see front matter © 2001 Elsevier Science Ltd. All rights reserved. PII: S0165-6147(00)01825-3

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(a)

(b)

From outside

II

VI

VII

IV

11-cis-retinal

I

III

Out

W265

II

V

K296

V

I

E122

V I

IV

All-trans

VII VI

In

III

VII

VIII

II

F115 III

IV A169

VIII VI

Fig. 1. (a) The crystal structure of rhodopsin. The seven transmembrane helices are shown with the following color coding: I, red; II, orange; III, yellow; IV, green; V, cyan; VI, dark blue; and VII, magenta. 11-cis-retinal is also shown in magenta. Helix VIII is shown in light pink, and all the nonhelical portions are shown in gray. Connections (black) have been drawn to roughly represent segments that are missing from the structure (i.e. four residues in intracellular loop 3 and six residues in the C-terminal tail). The yellow panels show the approximate boundaries of the hydrophobic core of the membrane. The high-resolution rhodopsin coordinates1 are from entry 1F88 in the Protein DataBank (PDB)48. All measurements and figures are based on the coordinates of molecule A in the crystal structure; molecular graphics and modeling were done using UCSF MidasPlus49 and its successor program UCSF Chimera (http://www.cgl.ucsf.edu/chimera/). (b) Conformational changes that occur during activation, as suggested by the data reviewed in the main text. The helices and cytoplasmic regions (residues 334–348 omitted for clarity) are shown from the cytoplasmic side with the same color coding as in (a). Arrows in the diagram indicate an outward (away from the bundle) movement of the cytoplasmic ends of III, VI and VII. A clockwise rotation of VI about its helical axis as viewed from the intracellular side might also occur8.

Activation model

Experiments in several laboratories, performed before the new structure of rhodopsin became available, produced a crude qualitative picture of the conformational changes that occur during receptor activation (Fig. 1b). In this scenario, the helical bundle blossoms open at its cytoplasmic end, exposing various regions for interaction with the G protein; the strongest evidence indicates activation-induced separation of helices III and VI. Overall movement of VI probably exceeds that of III, which is more constrained by its central position in the helix bundle. Other helices probably also adjust their positions upon activation as well. The activation model includes an outward (away from the bundle) movement of the cytoplasmic ends of III, VI and VII. A clockwise rotation of VI about its helical axis, as viewed from the IC side, might also occur8. The inner faces of II, III, VI and VII become more exposed, and the cytoplasmic ends of IV and V become less exposed. Crosslinking of the chromophore

An elegant recent experiment revealed a crosslink between retinal and light-activated rhodopsin that is strikingly inconsistent with the ground state observed in the crystal structure of rhodopsin9 (Fig. 2), suggesting that activation causes a significant rearrangement of rhodopsin’s helical bundle. Low temperatures were used to trap specific states of rhodopsin bound to a retinal analog with an http://tips.trends.com

Fig. 2. Differential retinal photocrosslinking in the dark and activated states of rhodopsin. The seven transmembrane helices are displayed as viewed from the extracellular side, with the color coding: I, red; II, orange; III, yellow; IV, green; V, cyan; VI, dark blue; and VII, magenta. 11-cis-retinal is shown in magenta, and a model of all-trans-retinal is shown in turquoise. A retinal analog was found to crosslink to W265 (light pink) in helix VI in the dark state of rhodopsin, but to A169 (light purple) in helix IV in the activated state9. A yellow dashed line shows a straight path from K296 (the site of retinal attachment) to A169, which in the observed structure passes through helix III. F115 and E122, found in previous crosslinking work11, are indicated in light gray.

activatable crosslinker in its ionone ring. Different crosslinks were formed in the receptor’s ground and activated states. In the former state, retinal was observed to crosslink to W265 (VI:16)9, exactly as would have been predicted by the crystal structure, in which W265 is packed against the ionone ring of retinal. (The numbers of amino acid positions refer to bovine rhodopsin; Baldwin numbers, such as VI:16, indicate positions in an alignment with many other GPCRs4.) In the activated state, the crosslink involved A169 (IV:19), which in the crystal structure faces outward from the bundle9. As was noted previously10, a computer-generated conversion of 11-cis-retinal to the all-trans form (Fig. 2) causes the end of the ionone ring to point towards IV, in a horizontal orientation near the level of A169; in the inactive state, however, A169 faces in the opposite direction. In the crystal structure, a straight line drawn from the covalent attachment site of retinal (K296; VII:11) to A169 (IV:19) passes through helix III. Thus, activation probably induces helix III to move away from this region and/or tilts or rotates helices IV and VII to open a path between K296 and A169. Results of an earlier study are also consistent with the idea that photo-isomerization causes the ionone ring of retinal to sweep from its position in the ground state to a position pointing towards helix IV (Ref. 11). It is likely that ground and activated states of rhodopsin were both present in these experiments because the retinal analog crosslinked not only to W265 (consistent with the ground state), but also with rather distant residues in helix III, including F115 (III:5), which is close to A169 (IV:19) (Fig. 2). Additional sites of crosslinking in III (A117, E122, W126 and S127; III:7, 12, 16 and 17) might reflect intermediate states or merely conformational

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Table 1. Bridges in rhodopsin α–Cα αc Residue paira Baldwin numbersb Cα distance (Å)

Categoryd

Refs

65–316

IC1–VIII

7.72

Permissive SS

13,19,21

110–187

III:0–EC2

5.46

Permissive SS

19

136–222

III:26–V:21

6.56

Inhibitory SS

14

136–225

III:26–V:24

7.58

Permissive SS

14

139–225

III:29–V:24

9.33

Permissive SS

14

140–222

III:30–V:21

9.20

Inhibitory SS

14

140–225

III:30–V:24

6.78

Permissive SS

14,19

138–251

III:28–VI:2

9.70

Inhibitory Zn2+

15

139–247

III:29–VI:–2

7.73

Inhibitory SS

8

139–248

III:29–VI:–1

8.51

Inhibitory SS

8,13

139–249

III:29–VI:0

11.67

Inhibitory SS

8

139–250

III:29–VI:1

10.39

Inhibitory SS

8,13

139–251

III:29–VI:2

8.33

Inhibitory SS

8

140–316

III:30–VIII

28.97

Light-only SS

22

198–276

V:–3–VI:27

15.23

Permissive SS

18

200–276

V:–1–VI:27

9.67

Permissive SS

18,20

204–276

V:3–VI:27

7.85

Permissive SS

18–20

246–312

VI:–3–VIII

10.01

Inhibitory SS

13

242–338

IC3–C-terminus

14.39

Permissive SS

17

245–338

VI:–4–C-terminus

20.20

Permissive SS

13

aBovine

rhodopsin. EC, extracellular; IC, intracellular. cThe α-carbon separation in angstroms for each residue pair in the rhodopsin crystal structure1. dBridges in rhodopsin can be classified as permissive (allowing activation), inhibitory (inhibiting activation), or light-only (forming only after light activation). SS refers to disulfide linkages between cysteine residues, and Zn2+ refers to bridges formed by ligation of Zn2+ ions by histidine residues. bAbbreviations:

flexibility within a state. A mixture of states in these experiments appears possible because the wavelength of light used to induce crosslinking caused some isomerization of the chromophore. A later study using a retinal analog locked in the 11-cis conformation to prevent activation showed crosslinking to W265 and L266 (VI:16 and 17)12, as would be predicted by the crystal structure. Interhelical bridges constrain conformational change

Before the crystal structure of rhodopsin was available, several laboratories constructed bridges between helices of rhodopsin to assess proximity between individual residues (Table 1 and Fig. 3). Although constructed without direct knowledge of the 3D structure of rhodopsin, several cysteine– cysteine disulfide bridges prevented light activation of rhodopsin (Table 1 and Fig. 3). Five of these bridges connected helices III and VI (Refs 8,13) near the cytoplasmic end of the helix bundle, two connected the cytoplasmic ends of helices III and V (Ref. 14), and one connected helix VI with helix VIII near the VII–VIII junction13. A different kind of bridge (a metal bridge in which Zn2+ is chelated by substituted histidine side-chains) connected the cytoplasmic ends of helices III and VI, and similarly prevented receptor activation15. The overall pattern http://tips.trends.com

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suggests that activation requires changes in the relative positions of III and VI, most likely a separation of their cytoplasmic ends (Fig. 1b). It is inferred from the inhibitory III–V crosslinks that activation requires changes in the relative positions of these helices without a large degree of separation, because nearby crosslinks between the same two helices permitted activation (Table 1 and Fig. 3). Because the crosslinks involving a residue higher up in helix V (222 but not 225) were inhibitory, helix III might become less tilted upon activation (Fig. 1b), that is, more perpendicular to the plane of the membrane, causing its end to protrude more into the cytoplasm. Inhibition of receptor function by the VI–VIII crosslink could indicate that helix VI moves away from the bundle more than VII and VIII do upon activation, or in a divergent direction (Fig. 1b). Indeed, activation does increase the accessibility of helix VII to the cytoplasm; light makes a stretch of amino acids near the VII–VIII junction accessible to an antibody16. Although no disulfides that lock rhodopsin into an active state were found, ten disulfides permitted activation (Table 1 and Fig. 3)13,14,17–21. Thus, activation does not require much relative movement of the positions connected by each permissive bridge. On the cytoplasmic side of the receptor, in addition to the three permissive III–V crosslinks noted above, two permissive crosslinks connected the IC loop 3 region to the C-terminal tail (residues 242–338 and 245–338); although the α-carbons of these pairs of residues are far apart (Table 1), it is imagined that flexibility of the C-terminal tail allows it to sample conformations that bring 338 closer to 242 and 245. Three helix V–VI disulfide bonds engineered on the EC side of rhodopsin permitted activation of rhodopsin (Fig. 3b)18–20. This suggests that the EC ends of helices V and VI need not separate from one another upon light activation. Also on the EC side, a ‘permissive’ native disulfide connects residue 110 in helix III to residue 187 in a β-strand in the cap of the receptor. One disulfide bond – connecting residues 140 (III:30) and 316 (VIII)22 – was found only after photoactivation of rhodopsin (Table 1 and Fig. 3b). This crosslink connected α-carbons that are 29 Å apart in the ground-state crystal, a distance much too great for connection by a cysteine– cysteine disulfide bond. One possibility is that light activation causes a significant conformational change that brings the two positions together. The 140–316 disulfide would probably prevent rhodopsin from activating transducin, however, because it would hinder access to cytoplasmic regions of the receptor thought to play important roles in coupling to the G protein23–25. It appears more likely that activation promoted formation of this disulfide by increasing the exposure and flexibility of the regions involved. Most of the α-carbon-to-α-carbon distances between residues (Table 1) are consistent with the ability of the substituted cysteines to crosslink. Although the distance between α-carbons of disulfide-bonded

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Fig. 3. Bridges in rhodopsin. The helices are shown with the following color coding: I, red; II, orange; III, yellow; IV, green; V, cyan; VI, dark blue; and VII, magenta. The bridges in Table 1 are shown as lines between the α-carbons of the residues involved, which are drawn here as small spheres. Permissive disulfide bridges are shown in orange, inhibitory disulfide bridges are shown in black, the inhibitory Zn2+ bridge is shown in green, and the light-only disulfide bridge is shown in yellow. (a) A side view of the entire rhodopsin structure. (b) (i) A view from the extracellular (EC) side including only the transmembrane helices and EC loop 2 [other EC loops, the N-terminus and intracellular (IC) regions are not shown]. (ii) A view from the cytoplasmic side, including only the helices (the EC and IC loops, the C-terminus and the bridges involving the C-terminus are not shown).

Site-directed spin labeling

cysteine residues is in the range of 3.8–6.8 Å (Ref. 26), a study of helix–helix interactions within a globular protein of known structure showed that disulfides formed readily between cysteines substituted for residues with α-carbon separations (in the crystal structure) of ∼12 Å or less27. In addition to the very long distance between residues 140 and 316, discussed above, three permissive disulfides in rhodopsin fall beyond this range (Table 1). Each involves a region (the EC end of helix V or the C-terminal tail) that might be relatively more flexible. http://tips.trends.com

The laboratories of Khorana and Hubbell have individually replaced, with a cysteine, almost every residue in the cytoplasmic loops, proximal C-terminal tail and adjacent helical regions of rhodopsin8,17,21,28–38. These cysteines allow incorporation of covalently attached compounds whose unpaired electrons can be probed spectroscopically. Site-directed spin labels can indicate whether the environment of a side-chain is aqueous or hydrophobic and whether it is buried; in addition, it is possible to determine approximate distances between pairs of spin labels in proteins39. Spin labeling studies provided much of the evidence underlying the prevailing model of rhodopsin activation (Fig. 1b). The model is in close agreement with the 3D distribution of positions that undergo significant shifts in mobility upon activation (Fig. 4). An increase in the mobility of a side-chain can be interpreted as an increase in its exposure on the surface of the protein because the mobility of a buried side-chain is restricted. Collectively, the mobility changes observed suggest that activation opens a cleft on the cytoplasmic side of the helix bundle30,31,33. More specifically, the postulated rigidbody tilt or translation of VI (Fig. 1b), moving its cytoplasmic end out from the bundle, would simultaneously increase exposure at the cytoplasmic end of III (Ref. 30), and decrease the exposure of some positions near the end of V (Ref. 31), exactly as observed (Fig. 4). The cytoplasmic end of III might move in the opposite direction, away from VI (Fig. 1b); this could produce the observed decrease in mobility of positions near the end of IV (Ref. 30). Mobility changes in the I–IC1–II region upon activation (not shown) were relatively minor34. Spin labels were also used to measure distances between pairs of residues in rhodopsin (Table 2)8,21. Spin–spin interactions can be detected for distances up to ∼25 Å (Ref. 39), and approximate separations can be derived from the strengths of the interactions. The shifts in distance ranges are all consistent with a rigid-body motion of VI relative to III, with a separation of their cytoplasmic ends and a rotation of VI about its axis (clockwise as viewed from the cytoplasm, Fig. 1b)8. It should be noted, however, that changes in these ranges do not necessarily reflect changes in distances between α-carbons because the measurements are determined from electron spins

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near the ends of relatively long (up to 10 Å), flexible side-chains. In addition, it is evident that a lightinduced separation detected by spin labels might not be necessary for activation; thus, residues 65 (IC1) and 316 (VIII) apparently move further apart upon activation21, but a disulfide bond between them was permissive for activation (Table 1 and Fig. 3b). All of the disulfides engineered between III and VI were inhibitory (Table 2 and Fig. 3), regardless of whether the separation between spin labels at the same pair of positions increased or decreased upon activation8.

Side view

Reactivity to thiol-specific reagents

From cytoplasm

Fig. 4. Spin-label mobility shifts upon receptor activation. The helices are shown with the following colour coding: I, red; II, orange; III, yellow; IV, green; V, cyan; VI, dark blue; and VII, magenta. Extracellular regions and residues 334–348 have been omitted for clarity. A line (black) has been drawn to roughly represent the four residues missing from intracellular loop 3 (IC3). The α-carbons at positions where a significant change in mobility was observed upon rhodopsin activation are drawn as spheres. Increases in mobility (pink spheres) were observed for spin labels at residues: 136, 138, 139 and 140 (III:26,28,29,30)30; 244 (IC3)31; 250, 251 and 253 (VI:1,2,4)31; and 313 and 316 in helix VIII (Ref. 33). Decreases in mobility (purple spheres) were observed for spin labels at residues: 147 and 149 (IC2)30; 153 and 154 (IV:3,4)30; and 227 and 231 (IC3)31.

Table 2. Distances determined with spin labelsa α–Cα αc Cα

Disulfided

Refs

11–19

7.72

Permissive

21

23–25

8.51

Inhibitory

8

15–20

15–20

11.67

Inhibitory

8

139–250

15–20

12–14

10.39

Inhibitory

8

139–251

12–14

23–25

8.33

Inhibitory

8

139–252

15–20

23–25

11.46

None

8

Residuesb Dark

Distance (Å) Light

65–316

7–13

139–248

12–14

139–249

aDistances

between spin labels engineered into rhodopsin have been determined in the dark and light-activated states. bBovine rhodopsin. cα-Carbon separation in angstroms for each residue pair in the rhodopsin crystal structure1. dDescribes whether a disulfide bond forms between cysteine residues at the same two positions and, if so, whether it is permissive or inhibitory for activation.

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Figure 5 maps, on the crystal structure, the effect of activation on the accessibility of individual cysteinesubstituted positions to thiol-specific reagents29,35,36. When these experiments were performed in the dark, observed patterns of reactivity (Fig. 5a) were highly consistent with the rhodopsin structure in the ‘off’ state. In general, residues facing outwards from the bundle and the most cytoplasmic residues were the most reactive; the nonreactive positions face other helices and are relatively high up in the transmembrane region. However, residues near the end of helix VIII were anomalously unreactive, perhaps because the adjacent palmitoylation sites (residues 322 and 323) occlude the region or restrict its conformational freedom. Changes in reactivity induced by light are again consistent with the idea that activation opens a cleft in the cytoplasmic side of the transmembrane bundle (Fig. 5b). In helix II, reactivities increased at positions facing helix III or the core of the helix bundle. Activation increased reactivities of residues in helix VI that face helix VII and the bundle core. Taken together, these results are consistent with the crosslinking and spin label studies. They suggest that activation opens a space within the cytoplasmic side of the helix bundle and induces VI to swing out from (or rotate relative to) the helix bundle. Future directions

The crystal structure of rhodopsin greatly increases opportunities for several kinds of experiments. Some of these will test the prevailing model of rhodopsin activation by creating constraining crosslinks between helices. Others will explore regions of the receptor that have not been previously subjected to the experimental approaches discussed above. Still others will use the rhodopsin structure as a guide for exploring the structure and function of other GPCRs. This review mentions only a few of the possibilities. So far, published interhelical crosslinks in rhodopsin have been confined to a few pairs of helices, located for the most part near the protein’s cytoplasmic face. By increasing the number of constraints and testing the function of multiply bridged mutants, it might be possible to define a minimal set of conformational changes required for activation. The recently reported ‘straitjacketed’

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Fig. 5. (a) Scanning accessibility of cysteine-specific reagents in darkstate rhodopsin mutants. The helices are shown with the following color coding: I, red; II, orange; III, yellow; IV, green; V, cyan; VI, dark blue; and VII, magenta. Extracellular (EC) regions and residues 334–348 have been omitted for clarity. A line (black) has been drawn to roughly represent the four residues missing from intracellular loop 3 (IC3). The α-carbons at scanned positions are drawn as spheres. Accessibility was measured by assessing reactivity to different reagents. In the IC1 region29, white spheres at residues 63 (I:26), 65 (I:28), 66–69 (IC1), 70 (II:1) and 73 (II:4) indicate high reactivity with the thiol-specific reagent 4,4′-dithiopyridine (4-PDS); light gray spheres indicate medium reactivity at residues 60 (I:23), 62 (I:25), 64 (I:27), 72 (II:3) and 74 (II:5); dark gray spheres indicate low reactivity at residues 61 (I:24) and 71 (II:2); and black spheres indicate no reactivity at residues 56–59 (I:19–22) and 75 (II:6). In the helix VIII region35, white spheres at residues 310 (VII:25), and at residues 312, 313 and 315 in helix VIII indicate high reactivity with 4-PDS; light gray spheres indicate medium reactivity at residues 308–309 (VII:23–24) and at residues 311, 314, 316 and 319 in helix VIII; dark gray spheres indicate low reactivity at residues 306–307 (VII:21–22); and black spheres indicate no reactivity at residues 317, 318, 320 and 321, all in helix VIII. Reactivity of cysteines to a large, rigid molecule, PyMPO-maleimide, was tested in helix VI (Ref. 36): white represents reactivity at 248–249 (VI:−1–0), 251–252 (VI:2–3), and 255 (VI:6); dark gray represents low reactivity at 256 (VI:7); and black represents lack of reactivity at residues 250 (VI:1) and 253–254 (VI:4–5). (b) Accessibility changes upon rhodopsin activation. EC regions and residues 334–348 have been omitted for clarity. A line has been drawn to roughly represent the four residues missing from IC3. The α-carbons at positions with increased (pink) or decreased (purple) accessibility upon activation are drawn as spheres. In the IC1 region29, the change in rate of dithiothreitol reduction of 4-PDS adducts was evaluated: pink spheres indicate an increase at residues 68 (IC1) and 70–72 (II:1–3); purple spheres indicate a decrease at residues 69 (IC1) and 73 (II:4). Positions in VI accessible to PyMPO-maleimide only upon activation36 are shown as pink spheres at residues 250 (VI:1) and 253 (VI:4).

Acknowledgements We thank David Farrens, Shaun Coughlin, Thomas Baranski, and members of the Bourne laboratory for useful discussions and for reading the manuscript. Molecular graphics images were produced using the MidasPlus and Chimera packages from the Computer Graphics Laboratory, University of California, San Francisco, CA, USA (supported by National Institutes of Health P41 RR-01081). This work was supported by National Institutes of Health grant GM-27800, awarded to H.R.B.

rhodopsin with engineered disulfides at residues 65–316, 140–225 and 204–276 (Ref. 19) was a first step in this direction. Bridges have also been ‘transferred’between different GPCRs. For example, a bridge in the β2-adrenoceptor between III and VI (III:28–VI:2), cognate to that shown in Fig. 3, similarly prevented ligand activation; a bridge at a similar position (III:26–VI:2) inactivated the parathyroid hormone receptor40. It is likely that other crosslinks will induce activation rather than prevent it. In two other GPCRs, an activating metal bridge was engineered between cognate residues in the EC third of helices III and VII (Refs 41,42). The analogous positions in rhodopsin might also form an activating bridge. It will be important to determine how the EC parts of other GPCRs differ from the elaborate lid over the retinal-binding pocket of rhodopsin (Fig. 1a). Such a lid seems unlikely for most GPCRs, whose ligands differ from retinal in that they interact rapidly and reversibly with their binding pockets. However, the 110–187 (III:0–EC2) disulfide of rhodopsin (Fig. 3b) is rather broadly conserved. Interestingly, analysis of a recent further refinement of the rhodopsin crystal structure43 reveals cavities near this region. The EC structures of other GPCRs might resemble those of rhodopsin somewhat, but with enough flexibility to allow rapid entry and exit of ligands. This region could be explored with cleverly designed EC-domain chimeras or by engineering permissive crosslinks into the known structure of rhodopsin and asking whether cognate crosslinks are tolerated in other GPCRs. http://tips.trends.com

Concluding remarks

The work of Palczewski et al.1 replaces more or less speculative structural models with a crystal structure at atomic resolution. The structure significantly strengthens the prevailing model of the molecular mechanism by which light activates rhodopsin; now we can see, in 3D, the striking agreement between restrictive crosslinks, site-directed spin labels, and relative accessibilities of positions in the IC loops and cytoplasmic ends of the helices. Importantly, the model is also consistent with a large body of mutational data from experiments with many receptors. These experiments show that disrupting interhelical contacts within a GPCR, particularly those involving helices III, VI and VII, frequently yields a constitutively active receptor44–47. This agreement also sets the stage for more stringent tests of the model and for extending it beyond the cytoplasmic regions, which have until now been the principal focus of such experiments. Questions and potentially informative experiments abound, not only with rhodopsin but also with other GPCRs. We do not know, of course, how long we shall have to wait for a 3D structure of rhodopsin in its light-activated form. Even when that structure is solved, however, biochemical experiments like those described here will help us to understand the transition of the receptor from its dark-adapted to its light-activated form. In turn, new structures and new experiments will gradually unfold a picture of how the large and ubiquitous family of GPCRs transmits signals across the plasma membrane.

Review

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