NMR structural comparison of the cytoplasmic juxtamembrane domains of G-protein-coupled CB1 and CB2 receptors in membrane mimetic dodecylphosphocholine micelles.

The fourth cytoplasmic domain, the so-called C-terminal juxtamembrane segment or helix VIII, has been identified in numerous G-protein-coupled receptors and exhibits unique functional characteristics. Efforts have been devoted to studying the juxtamembrane segment in order to understand the biological importance of the segment in G-protein activation of the cannabinoid CB1 and CB2 receptors. Recent biochemical data revealed that the CB1 C-terminal juxtamembrane peptide fragment CB1-(401-417) can directly activate the G-protein and also showed that the specificity of the signal transduction activation by the C-terminal juxtamembrane region is unique to the CB1 receptor but not to the CB2 receptor (Mukhopadhyay, S., and Howlett, A. C. (2001) Eur. J. Biochem. 268, 499-505). However, there is experimental work, not yet reported, on the conformational analyses and structural comparison between the respective helix VIII segments of the two receptors. In the present study, we have examined the conformational specificities of the cytoplasmic helical domains for both cannabinoid receptors. Three-dimensional structural features of two synthetic CB1 and CB2 peptides, CB1I397-G418 and CB2I298-K319, respectively, in membrane mimetic DPC micelles were studied using a combined high resolution NMR and computer modeling approach. Comparisons of the NMR-determined structures of the two peptides as well as their correspondent mutant peptides revealed their conformational properties and salt bridge dissimilarity, which might help us to understand the different structural roles of the fourth cytoplasmic helices in the function and regulation of CB1 and CB2 receptors.

apeutic applications, including antiemetics, appetite stimulants, analgesics, glaucoma treatment, and immune suppression (3,4). The CB 1 receptor is located in the central and peripheral nervous system (1,5,6), whereas the CB 2 receptor is distributed peripherally in non-neuronal tissues, particularly in immune cells (2,6). The CB 1 receptor interacts with the pertussis toxin-sensitive G i/o family of G-proteins to inhibit adenylate cyclase (7) and to regulate N-type Ca 2ϩ channels and inwardly rectifying K ϩ channels in the central nervous system (8 -10). The CB 2 receptor is expressed in high quantities in the human spleen and tonsils and exhibits 44% amino acid identity to the CB 1 receptor throughout the whole protein. The CB2 receptor is likely involved in the signal transduction processes in the immune system (6).
Studies have been carried out to understand the three-dimensional structure of the CB receptors and their mechanisms of action by using computer molecular modeling and NMR approaches (11)(12)(13)(14)(15)(16)(17). Bramblett et al. (11) first analyzed the secondary structure of the CB 1 receptor based upon calculated hydrophobicity and variability profiles to predict the regions of ␣-helicity and then predicted the three-dimensional structure model of the CB 1 receptor showing an extracellular N-terminal, seven transmembrane helical segments, three intracellular loops, and three extracellular loops, as well as a juxtamembrane helical C-terminal domain. Xie et al. (17) applied computer homology-based multiple sequence and conserved residue alignments to construct a human CB 2 three-dimensional structure model based on the recently available x-ray crystal structure of bovine rhodopsin (18), the first three-dimensional atomic structure in the GPCR family.
Efforts have also been devoted to investigating the CB receptor pharmacology and biological functions. In particular, attention has been focused on the C-terminal juxtamembrane domain in order to understand the biological importance of the domains in CB 1 and CB 2 activation. Mukhopadhyay et al. systematically investigated the biological functions of the CB 1 juxtamembrane region peptide CB 1 401-417 (19 -21). They concluded that the C-terminal juxtamembrane fragment peptide CB 1 401-417 can directly activate the G i protein and, in addition, the specificity of the C-terminal juxtamembrane region's affinity to G i and G o proteins is unique to the CB 1 receptor (19 -21) but not to the CB 2 receptor (21). On the other hand, Feng and Song (22) investigated the role of two cysteine residues in the C-terminal juxtamembrane region of human CB 2 with site-directed mutagenesis. They found that the C313A and C320A mutations markedly reduced functional coupling to adenylate cyclase but had no effect on ligand binding and agonist-induced receptor desensitization. Their results suggested that the conserved cysteine residues in the C-termi-nal juxtamembrane region play different roles among GPCRs. These early investigations on the juxtamembrane regions have underscored the biological importance of the fourth cytoplasmic domain and provided a tentative hypothesis on its roles in CB 1 and CB 2 receptor function. However, only limited structural information has been reported regarding the structural and conformational specificities of the C-terminal juxtamembrane domains of the cannabinoid receptors along the transmembrane region of the GPCRs.
In the present study, we presented our recent NMR studies and computer structural calculations of two C-terminal juxtamembrane or fourth cytoplasmic domains of CB 1 and CB 2 receptors, CB 1 I397-G418 and CB 2 I298-K319, bound to membrane mimetic DPC micelles. Both polypeptides show the stable ␣-helical secondary structure in the membrane-like environment. Structural comparisons of the two peptides are correlated with the structure-function relationship of C-terminal juxtamembrane domains. In addition, the structural properties of these two target peptides were examined against their correspondent mutant peptides. The results from the present study, in conjunction with published works (19 -21), support the hypothesis that the amphipathic helix nature of both juxtamembrane domains (or helix VIII fragments) and the salt bridge feature could potentially play significant roles in the function and regulation of the CB 1 and CB 2 cannabinoid receptors.

MATERIALS AND METHODS
Peptide Synthesis-Two peptides, CB 1 I397-G418 of the sequence IY-ALRSKDLRHAFRSMFPSCEG and CB 2 I298-K319 of the sequence IY-ALRSGEIRSSAHHCLAHWKK, were synthesized through solid-state phase synthesis at the biotechnology center of the University of Connecticut. Synthesis was performed on an Applied Biosystem 433A peptide synthesizer, using Fmoc (N-(9-fluorenyl)methoxycarbonyl) chemistry on p-hydroxymethylphenoxymethyl polystyrene resin at room temperature.
NMR Experiments-The NMR samples of CB 1 I397-G418, the CB1I397-G418(R401A) mutant, CB 2 I298-K319, and the CB2I298-K319(E305Q) mutant were prepared by adding solution of DPC-d38 (Cambridge Isotope Laboratories, Andover, MA) in sodium phosphate buffer (pH 5.7) containing 10% D 2 O (99.9%; Isotec, Inc.) to the powder polypeptides. To optimize the concentration of the DPC micelle for peptide binding, one-dimensional NMR spectroscopy experiments were performed to examine the peptide sample with various molar ratios of DPC-d38. The final samples were 2 mM in peptides and 200 mM in DPC-d38. All NMR spectra were recorded at 298 K on a Bruker DMX-600 spectrometer equipped with a 5-mm QXI probe with z-gradient. The two-dimensional, phase-sensitive NOESY (23) spectra were recorded with a mixing time of 100 ms. The TOCSY experiments were acquired according to standard pulse sequence (Malcom Levitt decoupling se-quence17) (24) with 70-and 90-ms spin-lock periods. All two-dimensional experiments (TOCSY, NOESY) used in peak assignments were recorded in a phase sensitive mode using time-proportional phase incrementation (TPPI) for quadrature detection in F1, with 128 transients, 512 complex points in the t1 dimension, and 4048 points in the t2 dimension. Data sets were processed on a SGI workstation utilizing XWIN-NMR software from Bruker.
Structural Calculations-Upon completion of the proton assignments (25), NOE inter-proton distance restraints were obtained from NOESY spectra and automatically allocated by using the Sybyl/DIANA program for close range, medium range, and long distance interactions, corresponding to upper limits of 2.5, 3.8, and 5.0 Å, respectively, based on the NOE peak volume. Pseudo-atoms were applied to stereospecificity-undistinguished protons in the methylene and methyl group. All calculations and graphic displays were performed on an Octane R12,000 IRIS SGI workstation using the Tripos/Sybyl 6.9.2 (26) molecular modeling package. Starting with the extended structure, Sybyl/ DIANA (26, 27) was carried out for initial structural calculation. 100 structures generated from DIANA were then subjected to extensive energy minimization using the Sybyl program (26) under the Kollman all-atom force field and Kollman charges to fold structures that were consistent with the available experimental distance constraints. Finally, 10 representative three-dimensional structures from the two peptides were selected with the lowest total energies and the criteria of no NOE violations Ͼ0.30 Å and a root mean square difference for bond deviations from ideality of Ͻ0.01 Å.

RESULTS
One-dimensional proton experiments of peptide CB 2 I298-K319 was carried out as a function of DPC concentrations in order to exam the peptide-micelle interaction and to achieve the best concentration of DPC micelles as illustrated in Fig. 1. Fig. 1A showed that the spectrum of the peptide in pure water (H 2 O/D 2 O, 90:10) displays sharp peaks with a narrow line shape, indicating a homogenous peptide solution. Upon the addition of DPC into the solution, the changes of chemical shifts and the increase in the peak line width were detected, revealing the presence of a conformational exchange of peptides in the micelle solution. At a 12.5 molar ratio of [DPC]/[CB 2 I298-K319] (Fig. 1D), the conformational exchange was observed, indicating a millisecond to microseconds time scale equilibrium between the free and micelle-bond states of peptide CB 2 I298-K319. The sharp peak shape in Fig Sequential Resonance Assignments-The proton NMR assignments were obtained from the two-dimensional 1 H-1 H TOCSY and 1 H-1 H NOESY spectra through the sequential dNN(i,iϩ1), d␣N(i,iϩ1), and d␤N(i,iϩ1) NOEs. The chemical shifts of the amide protons of the residues are dispersed over a range of 1 ppm. The two-dimensional 1 H-1 H NOESY spectra of both CB 1 and CB 2 fragments bound to the DPC micelles are given as examples in Fig. 2. The sequential chemical shift assignments were made by choosing the residues Ala and Tyr as the logical starts for the assignment process. The Tyr residue has typical cross peaks between the NH, ␣H, ␤H, and aromatic protons in the NOESY spectrum, and there is only one Tyr residue in each of the two synthetic peptides. For example, on the basis of the connection of the ␣H and ␤H peaks of preceding residue (iϩ1) from the ␤H␣H(CO)NH spin system with the ␣H and ␤H of adjacent residue (i) from the NH␣H␤H, the corresponding residues were identified. A similar analogy was applied to assign adjacent residues and repeated until all residues were assigned. Fig. 2 and Table I summarize the chemical shift assignments of proton resonance for both peptides.
NOE Interactions-NOESY spectra were used not only to determine the sequential assignments but also to obtain the NOE distance constraints and determine the peptide secondary structure. The NOESY spectrum of the peptide CB 1 I397-G418 in DPC micelles revealed an ␣-helical conformation on the basis of the observed typical helix-indicating NOE patterns of dNN(i,iϩ1), d␣N(i,iϩ3), and d␣␤(i,iϩ3) cross-peaks as shown in Fig. 3A. The NOE data showed that there are two helical segments formed, namely helix a from residues Ile 397 to Leu 400 and helix b from Ser 402 to Phe 413 in the target peptide CB 1 I397-G418. Additional NOE interactions in this peptide were detected between Ala 399 (␣H) and Leu 405 (NH), Ala 399 (␤H) and Leu 405 (NH), and Ala 399 (␣H) and Leu 405 (␥H). These NOE data provided the basis for the distance-constrained structural calculations for determining the relative orientation between helix a and helix b of the CB 1 C-terminal juxtamembrane peptide. Additionally, Our NMR data shows that only the d␣␦(i,iϩ1) NOE was observed for the residue Pro 414 in CB 1 I397-G418, indicating that this proline residue is in the trans conformation (25). The residues after proline in CB 1 I397-G418 appear to be in a random coil. On the other hand, Fig. 3B (mutant peptide) revealed different NOE patterns as depicted in the observed NOEs, i.e. d␣N(i,iϩ3), d␣N(i,iϩ4) and d␣␤(i,iϩ3), in comparison with the non-mutant peptide (Fig. 3A). The data indicated that the peptide CB 1 I397-G418(R401A) mutant, in which the residue Arg 401 was mutated to Ala, can form one continuous helical conformation ranging from Ile 397 to Phe 413 without a right angle bend at the fifth residue as the peptide CB 1 I397-G418.
The NOESY spectrum of peptide CB 2 I298-K319 in DPC micelles also supports an ␣-helical conformation with the typical NOE pattern of dNN(i,iϩ1), d␣N(i,iϩ3), and d␣␤(i,iϩ3) crosspeaks as shown in Fig. 3C. As with the respective CB 1 peptide fragment above, the Fig. 3C NOE patterns also indicated that two ␣-helical portions, a and b, are formed in the CB 2 I298-K319 peptide, showing that helix a comprises the residues Ile 298 to Leu 301 and that helix b includes the Ser 303 to Lys 319 residues. These two helices are connected at the Arg 302 residues. The two peptides CB 1 I397-G418 and CB 2 I298-K319 exhibit similar NOE interactions between the correspondent two helical segments as shown in Figs. 2 and 3, A and C. On the other hand, the helix b in the peptide CB 2 I298-K319 is longer than the one in the peptide CB 1 I397-G418. That is expected, because the residue proline in CB 1 I397-G418 terminates the helical conformation. Furthermore, Fig. 3D showed that the CB 2 I298-K319(E305Q) mutant gave a similar NOE patent, which indicated that the peptide CB 2 I298-K319(E305Q) mutant retains the similar secondary structure as the peptide CB 2 I298-K319. The secondary structures of four peptides (CB 1 I397-G418 and its mutant and CB 2 I298-K319 and its mutant) were further confirmed by the secondary shift analysis of the H␣ resonances (26,27), which is illustrated in Fig. 3.
Comparison of the NOESY spectra of the CB 1 I397-G418 and CB 2 I298-K319 peptides showed many similarities between the CB 1 and CB 2 peptide fragments. Within the respective a and b portion of each peptide, unique NOEs were observed between Ala 399 (␣H) and Leu 405 (NH), Ala 399 (␤H) and Leu 405 (NH), and Ala 399 (␣H) and Leu 405 (␥H) in peptide CB 1 I397-G418, whereas peptide CB 2 I298-K319 had NOEs between Ala 300 (NH) and Ile 306 (NH), Ala 300 (NH) and Ile 306 (␣H), and Ala 300 (NH) and Ile 306 (␥H). Based on the existence of helical structures in both peptides, the respective NOE patterns suggest that there may Three-dimensional Structures of the C-terminal Juxtamembrane Domains-NMR-derived constraints for the peptides CB 1 I397-G418 and CB 2 I298-K319 were used as input for structure calculations as implemented in the software Sybyl/DIANA package. Ten of the 100 best scored structures of both peptides were then refined through energy minimization calculations with the Sybyl program. Fig. 4 shows the backbone superposition of the 10 lowest energy structures with no experimental violation Ͼ0.35 Å for CB 1 I397-G418 (A) and CB 2 I298-K319 (B), respectively. The averaged pairwise root mean square values of the superimposed ten lowest energy structures were found to be 0.38 Å for backbone atoms and 1.52 Å for heavy atoms on the well defined region of 2-4 and 6 -17 residues in CB 1 I397-G418, and 0.40 Å for backbone atoms and 1.50 Å for all heavy atoms on the correspondent region of 2-4, 6 -21 residues in CB 2 I298-K319.
The topology and defined residues of the ␣-helices calculated using the NOE distance constraints were consistent with those determined by the NMR NOE analysis of secondary structures mentioned above and then showed a "L-shaped" turn at the fifth residue (Arg) between two helices a and b for both peptides. Figs. 3A and 4A show a break of the helical structure at the residue Arg 401 in CB 1 I397-G418. A similar turn was also observed at the residue Arg 302 for CB 2 I298-K319 (Figs. 3C and 4B). The respective breaks in each peptide resulted in the formation of two helical portions, a and b in which the two helical axes were almost perpendicular to each other (Fig. 4). The tight turns at Ala 399 -Lys 403 in CB 1 I397-G418 or Ala 300 - Ser 303 in CB 2 I298-K319 were also identified in the cannabinoid receptor three-dimensional molecular modeling structure using homology calculations (15,17). The measured distances of Ala 399 (␣H)/Leu 405 (NH), Ala 399 (␤H)/Leu 405 (NH), and Ala 399 (␣H)/Leu 405 (␥H) in CB 1 I397-G418 are 3.78, 4.75, and 4.13Å, respectively. The corresponding distances of Ala 300 (NH)/Ile 306 (NH), Ala 300 (NH)/Ile 306 (␣H), and Ala 300 (NH)/ Ile 306 (␥H) in CB 2 I298-K319 are 4.75, 3.39, and 3.62 Å, respectively. Helix a is a largely conserved region showing a high homology between CB 1 and CB 2 peptides. Fig. 5 displays the conformational similarity between the two peptide segments and the corresponding roles of the respective Arg residues in CB 1 I397-G418 and CB 2 I298-K319. Our results show that the side chain of Arg 401 in CB 1 I397-G418 (Fig. 5A) is in the extended conformation, whereas in the Arg 302 of CB 2 I298-K319 the side chain curves back and forms a salt bridge with the residue Glu 305 (Fig. 5B). The presence of a salt bridge in the peptide CB 2 I298-K319 is confirmed by the NOE interaction observed between Arg 302 (guanidine NH) andGlu 305 (␥H) in CB 2 I298-K319, but no NOE interaction between Arg 302 (guanidine NH) and Gln 305 (␥H) in the peptide CB 2 I298-K319(E305Q) mutant is observed.
Side Chain Orientations of Key Residues-The NMR-based computations of the structural conformations show that the cationic hydrophilic side chains, such as Lys 403 , Arg 406 , and Arg 410 in peptide CB 1 I397-G418 and Arg 307 , His 311 , Lys 318 in peptide CB 2 I298-K319 tend to orient on the same side of each correspondent helix. Our data also show that the orientation of these cationic side chain residues in the helix b portion (or C-terminal juxtamembrane domain) of the CB 1 or CB 2 receptors is opposite to helix a as shown in Fig. 5.
Cysteine residues are recognized as important residues in the C-terminal juxtamembrane regions of GPCRs. A number of studies (19,22,28,29) have indicated that the cysteines in the juxtamembrane region become palmitoylated in order to interact with the membrane. This palmitoylation/depalmitoylation process plays an important role in the functional coupling of GPCRs. Fig. 5 shows that the side chain of the cysteine residues, Cys 416 in CB 1 I397-G418 and Cys 313 in CB 2 I298-K319, are pointing toward the same direction toward the helical a segment or facing toward the membrane surface. It can be argued that cysteines in such a location may be palmitoylated to interact with the membrane bilayer. The result is also consistent with the crystal structure of the bovine rhodopsin (18), showing that two cysteine residues, Cys 322 and Cys 323 , at the end of the helix VIII of rhodopsin are palmitoylated. These palmitoylated residues serve to constrain the site of the helix VIII on the membrane surface (30). One the other hand, Cys 416 in CB 1 I397-G418 is in the random coil of the C-terminal peptide. DISCUSSION We have determined the three-dimensional structures of two peptides corresponding to the extended fourth cytoplasmic juxtamembrane domains within the CB 1 and CB 2 receptors by means of proton NMR measurements and NMR-derived, distance-constrained dynamics simulation. Our NMR results reveal that the C-terminal juxtamembrane domains of both CB 1 and CB 2 acquire helical conformations in membrane mimetic DPC micelles. These findings are consistent with the results of circular dichroism measurements of the peptides (15,19).
Our NMR NOE-constrained structural analyses of the extended CB 1 and CB 2 juxtamembrane peptides identify an Lshaped turn at the arginine residue in each peptide leading to two helical portions, a and b (Fig. 3, A and C, and Figs. 4 and 5). The results were confirmed by the observed NOEs between Ala 399 (␤H) and Leu 405 (NH), Ala 399 (␤H) and Leu 405 (␣H), and Ala 399 (␣H) and Leu 405 (␥H) for peptide CB 1 I397-G418, and by the NOEs between Ala 300 (NH) and Ile 306 (NH), Ala 300 (NH) and Ile 306 (␣H), and Ala 300 (NH) and Ile 306 (␥H) for peptide CB 2 I298-K319. Helix a in both peptides represents the highly conserved N-terminal region, which can be considered as the C-terminal region of the transmembrane domain helix VII in both cannabinoid receptors. Thus, if helix a is aligned along the helix VII axis, the L-shaped turn will then make the helix b portion, i.e. the C-terminal juxtamembrane domains (or helix VIII), almost parallel with the membrane surface. The connection between helices a and b corresponds to the turn between the transmembrane helix VII domain and the juxtamembrane helix VIII observed in the crystal structure of bovine rhodopsin (18). Such a right angle bend at the fifth residue of the CB 1 I397-G418 was also confirmed by our further NMR study of the peptide CB 1 I397-G418(R401A) mutant for which a continuing helical conformation was detected without the right angle bend at the fifth residue.
The homology/multiple sequence alignment (17) showed that part of the C-terminal juxtamembrane domains of both CB receptors include positively charged residues such as Arg, Lys, and His separated by hydrophobic residues. Such nonconsecutive cationic residues impart an amphipathic character to this helical peptide fragment. The structures of these two fragments were obtained from NMR NOE analyses (Figs. 2 and 3) and the constrained structural calculation (Fig. 4). It can be argued that such an amphipathic ␣-helical structure that includes a multiple positively charged motif is a requirement for its productive interaction of the receptor with G-proteins (19,31). The amphipathic characteristic of C-terminal juxtamembrane domain is by no means unique to the cannabinoid receptors. Other members of the GPCR superfamily such as rhodopsin and the ␤-adrenergic receptor, the human P2Y1 receptor, the dopamine receptor, the histamine receptor, the endothelin receptor, and others were found through sequence analysis to have similar amphipathic motifs in the corresponding region (32). Our results are thus congruent with a number of studies indicating that this region is responsible for the G-protein binding and activation (19,20,(33)(34)(35)(36).
The NOE-based structural calculations show that for the preferred conformers the hydrophilic cationic side chains of the residues Lys 403 , Arg 406 , and Arg 410 in CB 1 I397-G418 have the same orientations as those of the correspondent residues Arg 307 , His 311 , and Lys 318 in CB 2 I298-K319. As depicted in Figs. 4 and 5, these cationic residues in the CB 1 or CB 2 juxtamembrane domain are facing in an opposite direction of the helix a, part of C-terminal region of helix VII of receptors. This was determined on basis of the observed NOE patterns, e.g. the NOE interaction between the third Ala residue and the ninth Leu or Ile residues in two peptides. Therefore, our results suggest that the cationic side chains (Lys and Arg) in both peptides orient toward cytoplasmic regions; these findings are congruent with the x-ray crystal structure of the rhodopsin (18) and the three-dimensional homology model of CB 2 receptor (17), showing that the cationic hydrophilic residues face toward the cytoplasmic medium.
The helices VIII of the CB1 and CB2 receptor were demonstrated to present different roles in their respective abilities to interact with G-proteins concomitant with cAMP. In particular, the site mutation of Arg 401 of the CB 1 juxtamembrane fragment resulted in loss of affinity for the G-protein. One possible explanation is that the Arg residues in the helix VIII domains of the CB1 and CB2 receptors exist in different chemical environments. The NOE data analyses showed that Arg 302 in CB 2 I298-K319 exists in close proximity with Glu 305 , with which it may be forming a salt bridge that would serve to bridge the L-shaped structure of the two helical components (the a and b portions). On the other hand, our data with CB 1 I397-G418 did not allow us to identify a corresponding specific salt bridge that would serve to stabilize the corresponding two helical portions in the CB 1 model. Nevertheless, a potential candidate for such salt bridge interactions may occur between Asp 404 and His 407 in the CB1, which is supported by the relevant NOE pattern between Asp 404 (␤H) and His 407 (␤H). This postulation is supported by recent studies (19) demonstrating that the mutation of Arg 401 of the CB 1 juxtamembrane fragment results in loss of affinity for the G-protein. In addition, another cationic residue, Lys 403 , blocks the side chain of Arg 401 and prevents it from turning back to have salt bridge interaction with Asp 404 . Conversely, these two special cationic residues do not present in peptide CB 2 I298-K319. It appears that the helical conformation of the CB1 juxtamembrane domain made the important Arg 401 residue fully accessible for G-protein interactions, whereas the correspondent Arg 302 residue in the CB2 juxtamembrane was constrained by a salt bridge and became not available for G-protein.
In summary, our NMR analyses provide structural information on the CB 1 and CB 2 juxtamembrane segments. Such a structural determination of discrete domains is essential because of the intrinsic difficulties in the structure studies of seven transmembrane GPCRs. We have determined the secondary structures and conformations of the C-terminal juxtamembrane or the fourth cytoplasmic helix domains in the CB 1 and CB 2 receptors as well as the orientation of their positively charged residues. The NMR results show that both peptides exhibit ␣-helical conformations and that similarity exists in the relative orientation of their positively charged residues. In the juxtamembrane VIII domain of two CB receptors, the charged residues face toward the cytoplasmic medium, possessing an amphipathic helical property. In addition, our studies show that a different salt bridge may be formed in the correspondent CB1 or CB2 polypeptide fragments. Such conformational and structural differences may help to explain why the specificity of the signal transduction activation by the Cterminal juxtamembrane region is unique to the CB 1 receptor but not the CB 2 receptor. However, more detailed biological and biophysical studies are required to further elucidate the roles of the fourth cytoplasmic helix domain in G-protein binding. In addition, more vigorous studies of the relative conformations and orientations of the Helix VIII segments with respect to a membrane or other helix domains should be carried out to directly test the hypothesis in a much more extended peptide fragment or in an intact receptor system. This work is currently under way by using recombinant protein engineering and isotope-edited NMR spectroscopy. Ultimately, the structures observed would lead to direct biochemical tests that could serve to validate the proposed hypothesis.