Agonist-specific conformational changes in the yeast alpha-factor pheromone receptor.

The yeast alpha-factor pheromone receptor is a member of the G-protein-coupled receptor family. Limited trypsin digestion of yeast membranes was used to investigate ligand-induced conformational changes in this receptor. The agonist, alpha-factor, accelerated cleavage in the third intracellular loop, whereas the antagonist, desTrp1,Ala3-alpha-factor, reduced the cleavage rate. Thus, the enhanced accessibility of the third intracellular loop is specific to the agonist. alpha-Factor inhibited cleavage weakly at a second site near the cytoplasmic terminus of the seventh transmembrane helix, whereas the antagonist showed a stronger inhibition of cleavage at this site and at another site in the C-terminal domain of the receptor. The alpha-factor-induced conformational changes appeared to be inherent properties of the receptor, as they were retained in G-protein-deficient mutants. Moreover, a mutant receptor (ste2-L236H) that affects the third loop and is defective for G-protein coupling retained the ability to undergo the agonist-induced conformational changes. These results are consistent with a model in which G-protein activation is limited by the availability of specific contacts between the G protein and the third intracellular loop of the receptor. The antagonist appears to promote a distinct conformational state that differs from either the unoccupied or the agonist-occupied state.     

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.     

Identification of a Gs-protein coupling domain to the beta-adrenoceptor using site-specific synthetic peptides. Carboxyl terminus of Gs alpha is involved in coupling to beta-adrenoceptors

Competition between Gs-protein and the synthetic peptide, GSA 379-394, derived from the carboxyl-terminal region of the alpha s-subunit, led to complete inhibition of receptor-mediated adenylate cyclase activation in turkey erythrocyte membranes. Related peptides corresponding to the homologous carboxyl-terminal region of alpha t-, alpha il- or alpha o-subunits did not interfere with beta-receptor-Gs coupling. The direct coupling between Gs and adenylate cyclase was not influenced by any of these peptides. These results emphasize the important role of the carboxyl-terminus of G-protein alpha-subunits for the specific recognition of their corresponding receptors and for signal transduction.     

Two peptides from the alpha 2A-adrenergic receptor alter receptor G protein coupling by distinct mechanisms.

Peptides derived from various regions of the alpha 2A-adrenergic receptor (alpha 2A-AR) were used to study receptor-G protein interactions. Binding of the partial agonist [125I]-p-iodoclonidine and the full agonist [3H]bromoxidine (UK14,304) to membrane preparations from human platelet was potently reduced by peptides (12-14 amino acids) from the second cytoplasmic loop (A) and the C-terminal side of the third cytoplasmic loop (Q). Binding of the antagonist [3H]yohimbine was significantly less affected. Five other peptides had no significant effects on ligand binding at concentrations less than 100 microM. The IC50 values for peptides A and Q were 7 and 27 microM for [125I]-p-iodoclonidine binding at the platelet alpha 2A receptor, 15 and 71 microM for the neuroblastoma-glioma (NG108-15) alpha 2B receptor, and greater than 300 microM for yohimbine binding at both alpha 2A and alpha 2B receptors. Competition studies demonstrate that at concentrations of 100 microM, peptides A and Q reduce the affinity of bromoxidine for the platelet alpha 2A-AR and this effect was abolished in the presence of guanine nucleotide. Alpha 2A-AR-stimulated GTPase activity in platelet membranes was inhibited by peptide Q with an IC50 of 16 microM but A was inactive. These data suggest that both the second cytoplasmic loop and the C-terminal part of the third cytoplasmic loop of the alpha 2A-AR are important in the interaction between the alpha 2-AR and Gi protein. Peptide Q appears to destabilize the high affinity state of the alpha 2-AR by binding directly to Gi thus preventing it from coupling to the receptor under both binding and GTPase assay conditions. The peptide from the second cytoplasmic loop (A) also reduces high affinity agonist binding in a G protein-dependent manner but its interaction with receptor and G protein is distinct in that it does not prevent activation of the G protein. These results provide new information about regions of the alpha 2-adrenergic receptor involved in G protein coupling and high affinity agonist binding.     

beta-arrestin-dependent desensitization of luteinizing hormone/choriogonadotropin receptor is prevented by a synthetic peptide corresponding to the third intracellular loop of the receptor

Desensitization is a ubiquitous response of guanine nucleotide-binding protein-coupled receptors (GPCRs) characterized by the waning of effector activity despite continued presence of agonist. Binding of an arrestin to the activated, often phosphorylated GPCR triggers desensitization. We reported for the luteinizing hormone/choriogonadotropin receptor (LH/CG R) that beta-arrestin tightly bound to porcine ovarian follicular membranes mediates agonist-dependent desensitization of LH/CG R-stimulated adenylyl cyclase (AC) activity (Mukherjee, S., Palczewski, K., Gurevich, V. V., Benovic, J. L., Banga, J. P., and Hunzicker-Dunn, M. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 493-498). We now show that addition of a synthetic peptide corresponding to the entire third intracellular loop (3i) of the LH/CG R completely and specifically reverses desensitization of AC activity, with an ED50 of 10 microM but does not modulate basal, hCG-stimulated, or forskolin-stimulated AC activities. beta-Arrestin binds selectively to the 3i peptide coupled to activated Sepharose. Desensitization of LH/CG R-stimulated AC activity is rescued when the 3i peptide is preincubated with exogenous beta-arrestin. These results show that endogenous beta-arrestin participates in cell-free desensitization of agonist-dependent LH/CG R-stimulated AC activity in follicular membranes by interacting directly with the 3i loop of the receptor, thereby preventing Gs activation.     

Identification of a Gs activator region of the beta 2-adrenergic receptor that is autoregulated via protein kinase A-dependent phosphorylation.

We have localized a G protein activator region of the human beta 2-adrenergic receptor to region beta III-2 (from Arg259 to Lys273). The synthetic beta III-2, corresponding to the C-terminal end of the third cytoplasmic loop, activates Gs at nanomolar concentrations and weakly activates Gi. beta III-2 activates adenylyl cyclase at nanomolar concentrations in wild-type S49 lymphoma membranes, but not in membranes of unc mutant S49 cells, in which Gs is uncoupled from beta-adrenergic stimulation. Phosphorylation of beta III-2 by cAMP-dependent protein kinase A, which is involved in the desensitization of the beta-adrenergic receptor from Gs, drastically reduces the effect of beta III-2 on Gs while potentiating its action on Gi, resulting in a total loss of adenylyl cyclase-stimulating activity. These findings indicate that this receptor sequence is a multipotential G protein activator whose G protein specificity is regulated by protein kinase A.     

Involvement of the Glu724-Pro760 region of the dihydropyridine receptor II-III loop in skeletal muscle-type excitation-contraction coupling

Our previous study (El-Hayek, R., Antoniu, B., Wang, J. P., Hamilton, S. L., and Ikemoto, N. (1995) J. Biol. Chem. 270, 22116-22118) suggested the hypothesis that skeletal muscle-type excitation-contraction coupling is regulated by two domains (activating and blocking) of the II-III loop of the dihydropyridine receptor alpha1 subunit. We investigated this hypothesis by examining conformational changes in the ryanodine receptor induced by synthetic peptides and by transverse tubular system (T-tubule) depolarization. Peptide A, corresponding to the Thr671-Leu690 region, rapidly changed the ryanodine receptor conformation from a blocked state (low fluorescence of the conformational probe, methyl coumarin acetamide, attached specifically to the ryanodine receptor) to an activated state (high methyl coumarin acetamide fluorescence) as T-tubule depolarization did. Peptide C, corresponding to the Glu724-Pro760 region, blocked both conformational changes induced by peptide A and T-tubule depolarization. Its ability to block peptide A-induced and depolarization-induced activation was considerably impaired by replacing the portion of peptide C corresponding to the Phe725-Pro742 region of the loop with cardiac muscle-type sequence. These results are consistent with the model that depolarization-induced activation of excitation-contraction coupling and blocking/repriming are mediated by the peptide A region and the peptide C region (containing the critical Phe725-Pro742 sequence) of the II-III loop, respectively.     

Different domains in the third intracellular loop of the GLP-1 receptor are responsible for Galpha(s) and Galpha(i)/Galpha(o) activation

It has previously been shown that the GLP-1 receptor is primarily coupled to the adenylate cyclase pathway via activation of Galpha(s) proteins. Recent studies have shown that the third intracellular loop of the receptor is important in the stimulation of cAMP production. We have studied the effect of three synthetic peptide sequences derived from the third intracellular loop of the GLP-1 receptor on signal transduction in Rin m5F cell membranes. The whole third intracellular loop strongly stimulates both pertussis toxin and cholera toxin-sensitive G proteins, while the N-terminal half exclusively stimulates cholera toxin-sensitive G proteins and the C-terminal half only stimulates pertussis toxin-sensitive G-proteins as demonstrated by measurements of GTPase activity. These data confirm that the principal stimulatory G-protein interaction site resides in the third intracellular loop, but also suggest that the GLP-1 receptor is not only coupled to the Galpha(s) but also to the Galpha(i)/Galpha(o) type of G proteins and that distinct domains within the third intracellular loop are responsible for the activation of the different G-protein subfamilies.     

Identification of G-proteins coupling to the vasoactive intestinal peptide receptor VPAC(1) using immunoaffinity chromatography: evidence for precoupling.

VPAC(1) receptor subtype-specific G-protein interactions were identified using a strategy that exploits an essential initial signaling event, namely the functional and physical association of the receptor with G-protein. An immunoaffinity purification column was constructed using a previously characterized antibody that had been raised against the first extracellular loop of the VPAC(1) receptor. VPAC(1)/G-protein complexes were solubilized from membranes and copurified. Receptor and Galpha-proteins were detected in eluates using (125)I-VIP labeling and immunoblotting, respectively. Human VPAC(1) transfected in HEK293 cells couples to Gs but not Gi3, Gi1/2, or Gq. Rat VPAC(1) in brain membranes is coupled to Gs and Gi3. Rat VPAC(1) in lung membranes couples to Gs, Gi3, and Gq. Pretreatment of membranes with VIP increased the level of all G-proteins copurifying with VPAC(1). Immunoaffinity chromatography also revealed VPAC(1) receptor precoupling to G-protein in the absence of VIP pretreatment. This was confirmed using a cross-linking procedure to capture VIP receptor/G-protein complexes in the native membrane milieu prior to solubilization. Precoupling suggests that there is a significant basal level of VPAC(1) receptor activity especially in cells, such as some human malignant tumor cells, that express high levels of receptor.     

Activation of the rod G-protein Gt by the thrombin receptor (PAR1) expressed in Sf9 cells.

Functional coupling of the human thrombin receptor PAR1 (protease-activated receptor 1) with the retinal rod G-protein transducin (Gt, a member of the Gi family) was studied in a reconstituted system of membranes from Sf9 cells expressing the thrombin receptor and purified Gt from bovine rod outer segments. TRAP6-agonist-activated PAR1 interacts productively with the distant G-protein. Agonist-dependent Gt activation was measured using a real-time fluorimetric GTP[S]-binding assay and membranes from Sf9 cells. To characterize nucleotide-exchange catalysis by PAR1, we analyzed dependence on nucleotides, temperature and pH. Activation was inhibited by low GDP concentrations (IC50 = 5.2 +/- 1.5 microM at 5 microM GTP[S]), indicating that receptor-Gt coupling, followed by instantaneous GDP release, is rate limiting under the conditions (25 degrees C). Arrhenius plots of the temperature dependence reflect an apparent Ea of 60 +/- 3.5 kJ.mol-1. Evaluation of the pH/rate profiles of Gt activation indicates that the activating conformation of the receptor is determined by protonation of a titratable group with an apparent pKa of 6.4. This supports the idea that the active state of agonist-bound PAR1 depends on forced protonation, indicating possible analogies to the scheme established for rhodopsin.     

Distinct roles of the second and third cytoplasmic loops of bovine rhodopsin in G protein activation

In contrast to the extensive studies of light-induced conformational changes in rhodopsin, the cytoplasmic architecture of rhodopsin related to the G protein activation and the selective recognition of G protein subtype is still unclear. Here, we prepared a set of bovine rhodopsin mutants whose cytoplasmic loops were replaced by those of other ligand-binding receptors, and we compared their ability for G protein activation in order to obtain a clue to the roles of the second and third cytoplasmic loops of rhodopsin. The mutants bearing the third loop of four other G(o)-coupled receptors belonging to the rhodopsin superfamily showed significant G(o) activation, indicating that the third loop of rhodopsin possibly has a putative site(s) related to the interaction of G protein and that it is simply exchangeable with those of other G(o)-coupled receptors. The mutants bearing the second loop of other receptors, however, had little ability for G protein activation, suggesting that the second loop of rhodopsin contains a specific region essential for rhodopsin to be a G protein-activating form. Systematic chimeric and point mutational studies indicate that three amino acids (Glu(134), Val(138), and Cys(140)) in the N-terminal region of the second loop of rhodopsin are crucial for efficient G protein activation. These results suggest that the second and third cytoplasmic loops of bovine rhodopsin have distinct roles in G protein activation and subtype specificity.     

A mutation of the beta 2-adrenergic receptor impairs agonist activation of adenylyl cyclase without affecting high affinity agonist binding. Distinct molecular determinants of the receptor are involved in physical coupling to and functional activation of Gs

Activation of guanyl nucleotide regulatory proteins (G proteins) by hormones and neurotransmitters appears to require the formation of high affinity agonist-receptor-G protein ternary complexes. In the case of the beta 2-adrenergic receptor, multiple regions of the molecule have been implicated in coupling to the stimulatory G protein Gs. This finding raises the possibility that discrete regions of the receptor mediate ternary complex formation, whereas different loci may be involved in other aspects of G protein activation. To date, however, mutagenesis studies with the beta 2-adrenergic receptor have not clarified this question since mutant receptors with impaired abilities to activate Gs have generally possessed a diminished capacity to form the ternary complex as assessed in binding assays. We have expressed in a mammalian cell line a mutant beta 2-adrenergic receptor comprising a seven-amino acid deletion in the carboxyl-terminal region of its third cytoplasmic loop (D267-273), a region proposed to be critically involved in coupling to Gs. When tested with beta-adrenergic agonists, the maximal adenylyl cyclase response mediated by this mutant receptor was less than one-half of that seen with the wild-type receptor. Nevertheless, D267-273 exhibited high affinity agonist binding identical to that of the wild-type receptor. In addition, agonist-induced sequestration of the receptor, a property not mediated by Gs, was also normal. These findings indicate that the formation of high affinity agonist-receptor-Gs complexes is not sufficient to fully activate Gs. Instead, an additional stimulatory signal appears to be required from the receptor. Our data thereby suggest that the molecular determinants of the beta 2-adrenergic receptor involved in formation of the ternary complex are not identical to those that transmit the agonist-induced stimulatory signal to Gs.     

Domains for G-protein coupling in angiotensin II receptor type I: studies by site-directed mutagenesis.

To delineate domains essential for G-protein coupling in angiotensin II type 1 receptor (AT1), we mutated the receptor cDNA in the putative cytosolic regions and determined consequent changes in the effect of GTP analogs on angiotensin II (Ang II) binding and in inositol trisphosphate production in response to Ang II. Polar residues in targeted areas were replaced by small neutral residues. Mutations in the second cytosolic loop, carboxy terminal region of the third cytosolic loop or deletional mutation in the carboxyl terminal tail simultaneously abolished both the GTP-induced shift to the low affinity form and Ang II-induced stimulation of inositol trisphosphate production. These results suggest that polar residues in the second cytosolic loop, the carboxy terminal region of the third cytosolic loop, and the carboxy terminal cytosolic tail are important for G-protein coupling of AT1 receptor.     

Functional domains of membrane-bound human thrombomodulin. EGF-like domains four to six and the serine/threonine-rich domain are required for cofactor activity.

Thrombomodulin is an endothelial cell thrombin receptor that serves as a cofactor for thrombin-catalyzed activation of protein C. Structural requirements for thrombin binding and cofactor activity were studied by mutagenesis of recombinant human thrombomodulin expressed on COS-7 and CV-1 cells. Deletion of the fourth epidermal growth factor (EGF)-like domain abolished cofactor activity but did not affect thrombin binding. Deletion of either the fifth or the sixth EGF-like domain markedly reduced both thrombin binding affinity and cofactor activity. Thrombin binding sequences were also localized by assaying the ability of synthetic peptides derived from thrombomodulin to compete with diisopropyl fluorophosphate-inactivated 125I-thrombin binding to thrombomodulin. The two most active peptides corresponded to (a) the entire third loop of the fifth EGF-like domain (Kp = 85 +/- 6 microM) and (b) parts of the second and third loops of the sixth EGF-like domain (Kp = 117 +/- 9 microM). These data suggest that thrombin interacts with two discrete elements in thrombomodulin. Deletion of the Ser/Thr-rich domain dramatically decreased both thrombin binding affinity and cofactor activity and also prevented the formation of a high molecular weight thrombomodulin species containing chondroitin sulfate. Substitutions of this domain with polypeptide segments of decreasing length and devoid of glycosylation sites progressively decreased both cofactor activity and thrombin binding affinity. This correlation suggests that increased proximity of the membrane surface to the thrombin binding site may hinder efficient thrombin binding and the subsequent activation of protein C. Membrane-bound thrombomodulin therefore requires the Ser/Thr-rich domain as an important spacer, in addition to EGF-like domains 4-6, for efficient protein C activation.     

A single subunit (GB2) is required for G-protein activation by the heterodimeric GABA(B) receptor.

Although G-protein-coupled receptors (GPCRs) have been shown to assemble into functional homo or heteromers, the role of each protomer in G-protein activation is not known. Among the GPCRs, the gamma-aminobutyric acid (GABA) type B receptor (GABA(B)R) is the only one known so far that needs two subunits, GB1 and GB2, to function. The GB1 subunit contains the GABA binding site but is unable to activate G-proteins alone. In contrast the GB2 subunit, which does not bind GABA, has an heptahelical domain able to activate G-proteins when assembled into homodimers (Galvez, T., Duthey, B., Kniazeff, J., Blahos, J., Rovelli, G., Bettler, B., Prézeau, L., and Pin, J.-P. (2001) EMBO J. 20, 2152-2159). In the present study, we have examined the role of each subunit within the GB1-GB2 heteromer, in G-protein coupling. To that end, point mutations in the highly conserved third intracellular loop known to prevent G-protein activation of the related Ca-sensing or metabotropic glutamate receptors were introduced into GB1 and GB2. One mutation, L686P introduced in GB2 prevents the formation of a functional receptor, even though the heteromer reaches the cell surface, and even though the mutated subunit still associates with GB1 and increases GABA affinity on GB1. This was observed either in HEK293 cells where the activation of the G-protein was assessed by measurement of inositol phosphate accumulation, or in cultured neurons where the inhibition of the Ca(2+) channel current was measured. In contrast, the same mutation when introduced into GB1 does not modify the G-protein coupling properties of the heteromeric GABA(B) receptor either in HEK293 cells or in neurons. Accordingly, whereas in all GPCRs the same protein is responsible for both agonist binding and G-protein activation, these two functions are assumed by two distinct subunits in the GABA(B) heteromer: one subunit, GB1, binds the agonists whereas the other, GB2, activates the G-protein. This illustrates the importance of a single subunit for G-protein activation within a dimeric receptor.     

Identification of putative sites of interaction between the human formyl peptide receptor and G protein.

Wild-type and 35 mutant formyl peptide receptors (FPRs) were stably expressed in Chinese hamster ovary cells. All cell surface-expressed mutant receptors bound N-formyl peptide with similar affinities as wild-type FPR, suggesting that the mutations did not affect the ligand-binding site. G protein coupling was examined by quantitative analysis of N-formyl-methionyl-leucyl-phenylalanine-induced increase in binding of (35)S-labeled guanosine 5'-3-O-(thio)triphosphate (GTPgammaS) to membranes. The most prominent uncoupled FPR mutants were located in the N-terminal part of the second transmembrane domain (S63W and D71A) and the C-terminal interface of the third transmembrane domain (R123A and C124S/C126S). In addition, less pronounced uncoupling was detected with deletion mutations in the third cytoplasmic loop and in the cytoplasmic tail. Further analysis of some of the mutants that were judged to be uncoupled based on the [(35)S]GTPgammaS membrane-binding assay were found to transduce a signal, as evidenced by intracellular calcium mobilization and activation of p42/44 MAPK. Thus, these single point mutations in FPR did not completely abolish the interaction with G protein, emphasizing that the coupling site is coordinated by several different regions of the receptor. Mutations located in the putative fifth and sixth transmembrane domains near the N- and C-terminal parts of the third cytoplasmic loop did not result in uncoupling. These regions have previously been shown to be critical for G protein coupling to many other G protein-coupled receptors. Thus, FPR appears to have a G protein-interacting site distinct from the adrenergic receptors, the muscarinic receptors, and the angiotensin receptors.     

Coupling of dopamine receptors to G proteins: studies with chimeric D2/D3 dopamine receptors

D₂ and D₃ dopamine receptors belong to the superfamily of G protein-coupled receptors; they share a high degree of homology and are structurally similar. However, they differ from each other in their second messenger coupling properties. Previously, we have studied the differential coupling of these receptors to G proteins and found that while D₂ receptor couples only to inhibitory G proteins, D₃ receptor couples also to a stimulatory G protein, Gs. We aimed to investigate the molecular basis of these differences and to determine which domains in the receptor control its coupling to G proteins. For this purpose four chimeras were constructed, each composed of different segments of the original D₂ and D₃ receptors. We have demonstrated that chimeras with a third cytoplasmic loop of D₂ receptor couple to Gi protein in a pattern characteristic of D₂ receptor. On the other hand chimeras containing a third cytoplasmic loop of D₃ receptor have coupling characteristics like those of D₃ receptor, and they couple also to Gs protein. These findings demonstrate that the third cytoplasmic loop determines and accounts for the coupling of dopamine receptors D₂ and D₃ to G proteins.     

The molecular determinants in the serpentine type receptors,responsible for its functional coupling with the heterotrimeric G-protein

In the review, data of the literature and own results on the functional coupling between the serpentine type receptors and the heterotrimeric G-proteins are analyzed and summarized. The role of cytoplasmic loops and C-tail domain of the receptors in interaction with G-protein alpha-subunits of different types is discussed. On the basis of theoretical analysis it is shown that the second cytoplasmic loop and the proximal to the membrane segments of the third cytoplasmic loop, containing the main G-protein-coupled molecular determinants, have the cationic nature and can form the helical structures. A molecular model of signal transduction from the receptor to G-protein, based on the electrostatic interactions between the cytoplasmic loops of receptors and receptor-binding regions of G-proteins, is developed.     

The conformation of the cytoplasmic helix 8 of the CB1 cannabinoid receptor using NMR and circular dichroism

The cytoplasmic helix domain (fourth cytoplasmic loop, helix 8) of numerous GPCRs such as rhodopsin and the beta-adrenergic receptor exhibits unique structural and functional characteristics. Computational models also predict the existence of such a structural motif within the CB1 cannabinoid receptor, another member of the G-protein coupled receptor superfamily. To gain insights into the conformational properties of this GPCR component, a peptide corresponding to helix 8 of the CB1 receptor with a small contiguous segment from transmembrane helix 7 (TM7) was chemically synthesized and its secondary structure determined by circular dichroism (CD) and solution NMR spectroscopy. Our studies in DPC and SDS micelles revealed significant alpha-helical structure while in an aqueous medium, the peptide exhibited a random coil configuration. The relative orientation of helix 8 within the CB1 receptor was obtained from intermolecular 31P-1H and 1H-1H NOE measurements. Our results suggest that in the presence of an amphipathic membrane environment, helix 8 assumes an alpha helical structure with an orientation parallel to the phospholipid membrane surface and perpendicular to TM7. In this model, positively charged side chains interact with the lipid headgroups while the other polar side chains face the aqueous region. The above observations may be relevant to the activation/deactivation of the CB1 receptor.     

Mutation of the fourth cytoplasmic loop of rhodopsin affects binding of transducin and peptides derived from the carboxyl-terminal sequences of transducin alpha and gamma subunits

The role of the putative fourth cytoplasmic loop of rhodopsin in the binding and catalytic activation of the heterotrimeric G protein, transducin (G(t)), is not well defined. We developed a novel assay to measure the ability of G(t), or G(t)-derived peptides, to inhibit the photoregeneration of rhodopsin from its active metarhodopsin II state. We show that a peptide corresponding to residues 340-350 of the alpha subunit of G(t), or a cysteinyl-thioetherfarnesyl peptide corresponding to residues 50-71 of the gamma subunit of G(t), are able to interact with metarhodopsin II and inhibit its photoconversion to rhodopsin. Alteration of the amino acid sequence of either peptide, or removal of the farnesyl group from the gamma-derived peptide, prevents inhibition. Mutation of the amino-terminal region of the fourth cytoplasmic loop of rhodopsin affects interaction with G(t) (Marin, E. P., Krishna, A. G., Zvyaga T. A., Isele, J., Siebert, F., and Sakmar, T. P. (2000) J. Biol. Chem. 275, 1930-1936). Here, we provide evidence that this segment of rhodopsin interacts with the carboxyl-terminal peptide of the alpha subunit of G(t). We propose that the amino-terminal region of the fourth cytoplasmic loop of rhodopsin is part of the binding site for the carboxyl terminus of the alpha subunit of G(t) and plays a role in the regulation of betagamma subunit binding.