Role of the third intracellular loop of the angiotensin II receptor subtype AT2 in ligand-receptor interaction

Angiotensin II (Ang II) receptor subtypes AT1 and AT2 share 34% overall homology, but the least homology is in their third intracellular loop (3rd ICL). In an attempt to elucidate the role of the 3rd ICL in determining the similarities and differences in the functions of the AT1 and the AT2 receptors, we generated a chimeric receptor in which the 3rd ICL of the AT2 receptor was replaced with that of the AT1 receptor. Ligand-binding properties and signaling properties of this receptor were assayed by expressing this receptor in Xenopus oocytes. Ligand-binding studies using [125I-Sar1-Ile8] Ang II, a peptidic ligand that binds both the AT1 and the AT2 receptor subtypes, and 125I-CGP42112A, a peptidic ligand that is specific for the AT2 receptor, showed that the chimeric receptor has lost affinity to both ligands. However, IP3 levels of the oocytes expressing the chimeric receptor were comparable to the IP3 levels of the oocytes expressing the AT1 receptor, suggesting that the chimeric receptors could couple to phospholipase C pathway in response to Ang II. We have shown previously that the nature of the amino acid present in the position 215 located in the fifth transmembrane domain (TMD) of the AT2 receptor plays an important role in determining its affinity to different ligands. Our results from the ligand-binding studies of the chimeric receptor further support the idea that the structural organization of the region spanning the 5th TMD and the 3rd ICL of the AT2 receptor has an important role in determining the ligand-binding properties of this receptor.     

Role of the second extracellular loop of human C3a receptor in agonist binding and receptor function

The C3a anaphylatoxin receptor (C3aR) is a G protein-coupled receptor with an unusually large second extracellular loop (e2 loop, approximately 172 amino acids). To determine the function of this unique structure, chimeric and deletion mutants were prepared and analyzed in transfected RBL-2H3 cells. Whereas replacement of the C3aR N-terminal segment with that from the human C5a receptor had minimal effect on C3a binding, substitution of the e2 loop with a smaller e2 loop from the C5a receptor (C5aR) abolished binding of 125I-C3a and C3a-stimulated calcium mobilization. However, as much as 65% of the e2 loop sequence (amino acids 198-308) may be removed without affecting C3a binding or calcium responses. The e2 loop sequences adjacent to the transmembrane domains contain multiple aspartate residues and are found to play an important role in C3a binding based on deletion mutagenesis. Replacement of five aspartate residues in the e2 loop with lysyl residues significantly compromised both the binding and functional capabilities of the C3a receptor mediated by intact C3a or by two C3a analog peptides. These data suggest a two-site C3a-C3aR interaction model similar to that established for C5a/C5aR. The anionic residues near the N and C termini of the C3aR e2 loop constitute a non-effector secondary interaction site with cationic residues in the C-terminal helical region of C3a, whereas the C3a C-terminal sequence LGLAR engages the primary effector site in C3aR.     

Conformational features of a synthetic model of the first extracellular loop of the angiotensin II AT1A receptor.

The angiotensin II AT1A receptor belongs to the G-protein coupled receptors (GPCRs). Like other membrane proteins, GPCRs are not easily amenable to direct structure determination by the currently available methods. The peptide encompassing the putative first extracellular loop of AT1A (residues Thr88-Leu100, el1) has been synthesized along with a cyclic model where the linear peptide has been covalently linked to a template designed to keep the distance between the peptide termini as expected in the receptor. The conformational features of the two molecules have been studied using circular dichroism and NMR techniques. The region W94PFG97 forms a type-II beta-turn and undergoes a Trp-Pro peptide bond cis-trans isomerization in both peptides confirming that these characteristics are intrinsic to el1. In addition, the presence of the spacer seems to modulate the flexibility of the peptide.     

Identification of residues important for ligand binding of thromboxane A2 receptor in the second extracellular loop using the NMR experiment-guided mutagenesis approach

The second extracellular loop (eLP2) of the thromboxane A(2) receptor (TP) had been proposed to be involved in ligand binding. Through two-dimensional (1)H NMR experiments, the overall three-dimensional structure of a constrained synthetic peptide mimicking the eLP2 had been determined by our group (Ruan, K.-H., So, S.-P., Wu, J., Li, D., Huang, A., and Kung, J. (2001) Biochemistry 40, 275-280). To further identify the residues involved in ligand binding, a TP receptor antagonist, SQ29,548 was used to interact with the synthetic peptide. High resolution two-dimensional (1)H NMR experiments, NOESY, and TOCSY were performed for the peptide, SQ29,548, and peptide with SQ29,548, respectively. Through completed (1)H NMR assignment and by comparing the different spectra, extra peaks were observed on the NOESY spectrum of the peptide with SQ29,548, which implied the contacts between residues of eLP2 at Val(176), Leu(185), Thr(186), and Leu(187) with SQ29,548 at position H2, H7, and H8. Site-directed mutagenesis was used to confirm the possible ligand-binding sites on native human TP receptor. Each of the four residues was mutated to the residues either in the same group, with different structure or different charged. The mutated receptors were then tested for their ligand binding activity. The receptor with V176L mutant retained binding activity to SQ29,548. All other mutations resulted in decreased or lost binding activity to SQ29,548. These mutagenesis results supported the prediction from NMR experiments in which Val(176), Leu(185), Thr(186), and Leu(187) are the possible residues involved in ligand binding. This information facilitates the understanding of the molecular mechanism of thromboxane A(2) binding to the important receptor and its signal transduction.     

Role of the transmembrane domain 4/extracellular loop 2 junction of the human gonadotropin-releasing hormone receptor in ligand binding and receptor conformational selection

Recent crystal structures of G protein-coupled receptors (GPCRs) show the remarkable structural diversity of extracellular loop 2 (ECL2), implying its potential role in ligand binding and ligand-induced receptor conformational selectivity. Here we have applied molecular modeling and mutagenesis studies to the TM4/ECL2 junction (residues Pro(174(4.59))-Met(180(4.66))) of the human gonadotropin-releasing hormone (GnRH) receptor, which uniquely has one functional type of receptor but two endogenous ligands in humans. We suggest that the above residues assume an α-helical extension of TM4 in which the side chains of Gln(174(4.60)) and Phe(178(4.64)) face toward the central ligand binding pocket to make H-bond and aromatic contacts with pGlu(1) and Trp(3) of both GnRH I and GnRH II, respectively. The interaction between the side chains of Phe(178(4.64)) of the receptor and Trp(3) of the GnRHs was supported by reciprocal mutations of the interacting residues. Interestingly, alanine mutations of Leu(175(4.61)), Ile(177(4.63)), and Met(180(4.66)) decreased mutant receptor affinity for GnRH I but, in contrast, increased affinity for GnRH II. This suggests that these residues make intramolecular or intermolecular contacts with residues of transmembrane (TM) domain 3, TM5, or the phospholipid bilayer, which couple the ligand structure to specific receptor conformational switches. The marked decrease in signaling efficacy of I177A and F178A also indicates that IIe(177(4.63)) and Phe(178(4.64)) are important in stabilizing receptor-active conformations. These findings suggest that the TM4/ECL2 junction is crucial for peptide ligand binding and, consequently, for ligand-induced receptor conformational selection.     

Distinct second extracellular loop structures of the brain cannabinoid CB(1) receptor: implication in ligand binding and receptor function

The G-protein-coupled receptor (GPCR) second extracellular loop (E2) is known to play an important role in receptor structure and function. The brain cannabinoid (CB(1)) receptor is unique in that it lacks the interloop E2 disulfide linkage to the transmembrane (TM) helical bundle, a characteristic of many GPCRs. Recent mutation studies of the CB(1) receptor, however, suggest the presence of an alternative intraloop disulfide bond between two E2 Cys residues. Considering the oxidation state of these Cys residues, we determine the molecular structures of the 17-residue E2 in the dithiol form (E2(dithiol)) and in the disulfide form (E2(disulfide)) of the CB(1) receptor in a fully hydrated 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine bilayer, using a combination of simulated annealing and molecular dynamics simulation approaches. We characterize the CB(1) receptor models with these two E2 forms, CB(1)(E2(dithiol)) and CB(1)(E2(disulfide)), by analyzing interaction energy, contact number, core crevice, and cross correlation. The results show that the distinct E2 structures interact differently with the TM helical bundle and uniquely modify the TM helical topology, suggesting that E2 of the CB(1) receptor plays a critical role in stabilizing receptor structure, regulating ligand binding, and ultimately modulating receptor activation. Further studies on the role of E2 of the CB(1) receptor are warranted, particularly comparisons of the ligand-bound form with the present ligand-free form.     

Differential determinants for peptide and non-peptidyl ligand binding to the motilin receptor. Critical role of second extracellular loop for peptide binding and action.

The predicted second extracellular loop domain of the motilin receptor is of particular interest because it is a region that is quite distinct from the analogous regions in other family members that are most closely related and because the initial report of the photoaffinity labeling of a domain of this receptor included this region (Coulie, B. J., Matsuura, B., Dong, M., Hadac, E. M., Pinon, D. I., Feighner, S. D., Howard, A. D., and Miller, L. J. (2001) J. Biol. Chem. 276, 35518-35522). In the current work, motilin receptor constructs were prepared that included sequential deletions ranging from single residues to twelve amino acid segments throughout this 67 amino acid domain. Each construct was expressed in COS cells and characterized for motilin radioligand binding and motilin-stimulated intracellular calcium responses. The only segments that had negative impact on motilin binding and biological activity included deletion constructs DeltaCys(235), Delta179-182, and Delta241-246. Cys(235) is likely involved in the highly conserved and functionally important disulfide bond linking the first and second loops of G protein-coupled receptors. Alanine replacements for each of the amino acid residues in the other two segments revealed that the perimembranous residues at both ends of this loop, Val(179) and Leu(245) and Arg(246), were responsible for the negative impact on motilin binding and biological activity. Of note, these mutants responded normally to the non-peptidyl agonist, erythromycin. These data support important functional roles for both amino-terminal and carboxyl-terminal perimembranous regions of the second loop for responses to the natural agonist peptide, while supporting independent determinants for action of a non-peptidyl agonist ligand.     

Role of extracellular disulfide-bonded cysteines in the ligand binding function of the beta 2-adrenergic receptor

Evidence is presented for a role of disulfide bridging in forming the ligand binding site of the beta 2-adrenergic receptor (beta AR). The presence of disulfide bonds at the ligand binding site is indicated by "competitive" inhibition by dithiothreitol (DTT) in radioligand binding assays, by specific protection by beta-adrenergic ligands of these effects, and by the requirement of disulfide reduction for limit proteolysis of affinity ligand labeled receptor. The kinetics of binding inhibition by DTT suggest at least two pairs of disulfide-bonded cysteines essential for normal binding. Through site-directed mutagenesis, we indeed were able to identify four cysteines which are critical for normal ligand binding affinities and for the proper expression of functional beta AR at the cell surface. Unexpectedly, the four cysteines required for normal ligand binding are not those located within the hydrophobic transmembrane domains of the receptor (where ligand binding is presumed to occur) but lie in the extracellular hydrophilic loops connecting these transmembrane segments. These findings indicate that, in addition to the well-documented involvement of the membrane-spanning domains of the receptor in ligand binding, there is an important and previously unsuspected role of the hydrophilic extracellular domains in forming the ligand binding site.     

The unique extracellular disulfide loop of the glycine receptor is a principal ligand binding element.

A loop structure, formed by the putative disulfide bridging of Cys198 and Cys209, is a principal element of the ligand binding site in the glycine receptor (GlyR). Disruption of the loop's tertiary structure by Ser mutations of these Cys residues either prevented receptor assembly on the cell surface, or created receptors unable to be activated by agonists or to bind the competitive antagonist, strychnine. Mutation of residues Lys200, Tyr202 and Thr204 within this loop reduced agonist binding and channel activation sensitivities by up to 55-, 520- and 190-fold, respectively, without altering maximal current sizes, and mutations of Lys200 and Tyr202 abolished strychnine binding to the receptor. Removal of the hydroxyl moiety from Tyr202 by mutation to Phe profoundly reduced agonist sensitivity, whilst removal of the benzene ring abolished strychnine binding, thus demonstrating that Tyr202 is crucial for both agonist and antagonist binding to the GlyR. Tyr202 also influences receptor assembly on the cell surface, with only large chain substitutions (Phe, Leu and Arg, but not Thr, Ser and Ala) forming functional receptors. Our data demonstrate the presence of a second ligand binding site in the GlyR, consistent with the three-loop model of ligand binding to the ligand-gated ion channel superfamily.     

The angiotensin II AT2 receptor is an AT1 receptor antagonist

The vasopressor angiotensin II activates AT(1) and AT(2) receptors. Most of the known in vivo effects of angiotensin II are mediated by AT(1) receptors while the biological functions of AT(2) receptors are less clear. We report here that the AT(2) receptor binds directly to the AT(1) receptor and thereby antagonizes the function of the AT(1) receptor. The AT(1)-specific antagonism of the AT(2) receptor was independent of AT(2) receptor activation and signaling, and it was effective on different cells and on human myometrial biopsies with AT(1)/AT(2) receptor expression. Thus, the AT(2) receptor is the first identified example of a G-protein-coupled receptor which acts as a receptor-specific antagonist.     

A role for transmembrane domains V and VI in ligand binding and maturation of the angiotensin II AT1 receptor

Several studies have proposed that angiotensin II (Ang II) binds to its receptor AT1 through interactions with residues in helices V and VI, suggesting that the distance between these helices is crucial for ligand binding. Based on a 3D model of AT1 in which the C-terminus of Ang II is docked, we identified the hydrophobic residues of TM V and VI pointing towards the external face of the helices, which may play a role in the structure of the binding pocket and in the structural integrity of the receptor. We performed a systematic mutagenesis study of these residues and examined the binding, localization, maturation, and dimerization of the mutated receptors. We found that mutations of hydrophobic residues to alanine in helix V do not alter binding, whereas mutations to glutamate lead to loss of binding without a loss in cell surface expression, suggesting that the external face of helix V may not directly participate in binding, but may rather contribute to the structure of the binding pocket. In contrast, mutations of hydrophobic residues to glutamate in helix VI lead to a loss in cell surface expression, suggesting that the external surface of helix VI plays a structural role and ensures correct folding of the receptor.     

Exploring deltorphin II binding to the third extracellular loop of the delta-opioid receptor

The third extracellular loop of the human delta-opioid receptor (hDOR) is known to play an important role in the binding of delta-selective ligands. In particular, mutation of three amino acids (Trp(284), Val(296), and Val(297)) to alanine significantly diminished delta-opioid receptor affinity for delta-selective ligands. To assess the changes in conformation accompanying binding of the endogenous opioid peptide deltorphin II to the delta-opioid receptor at both the receptor and ligand levels as well as to determine points of contact between the two, an in-depth spectroscopic study that addressed these points was initiated. Fragments of the delta-opioid receptor of variable length and containing residues in the third extracellular loop were synthesized and studied by NMR and CD spectroscopy in a membrane-mimetic milieu. The receptor peptides examined included hDOR-(279-299), hDOR-(283-299), hDOR-(281-297), and hDOR-(283-297). A helical conformation was observed for the longest receptor fragment between Val(283) and Arg(291), whereas a nascent helix occurred in a similar region for hDOR-(281-297). Further removal of N-terminal residues Val(281) and Ile(282) abolished helical conformation completely. Binding of the delta-selective ligand deltorphin II to hDOR-(279-299) destabilized the helix at the receptor peptide N terminus. Dramatic changes in the alpha-proton chemical shifts for Trp(284) and Leu(286) in hDOR-(279-299) also accompanied this loss of helical conformation. Large upfield displacement of alpha-proton chemical shifts was observed for Leu(295), Val(296), and Val(297) in hDOR-(279-299) following its interaction with deltorphin II, thus identifying a gain in beta-conformation at the receptor peptide C terminus. Similar changes did not occur for the shorter peptide hDOR(281-297). A hypothesis describing the conformational events accompanying selective deltorphin II binding to the delta-opioid receptor is presented.     

Trifluoroethanol and binding to model membranes stabilize a predicted turn in a peptide corresponding to the first extracellular loop of the angiotensin II AT(1A) receptor

Deuterium oxide solutions of a triple-helical polysaccharide schizophyllan, undergoing an order-disorder transition centered around 17 degrees C, were studied by the time-domain reflectometry (TDR) to obtain dielectric dispersions in the solution and frozen states. In the solution state, the dispersion below the transition temperature is resolved in three dispersions (relaxation times at 0 degrees C) ascribed to side chain glucose residue (1; 102 ns), structured water (s; 2.0 ns) and bulk water (h), respectively, from low to high frequencies. Bulk water is divided into slow water (h2; 0.04 ns) and free or pure water (h1; 0.02 ns). Above the transition temperature structured water almost disappears and is compensated by slow water. Structured water is similar to bound water for proteins but different from it because of this transition behavior. Another dispersion (l) seen at the lowest frequency is assigned to the rotation of side-chain glucose residue coupled with hydrated water. Parts of this dispersion and structured water are suggested to constitute bound water. In the frozen state were observed a major dispersion (h; 0.14 ns) and a minor one (m; 28 ns), which were ascribed to considerably mobile and less mobile waters. They are similar to but not exactly the same as that for unfreezable water in bovine serum albumin solutions argued by Miura et al. (Biopolymers, 1995, Vol. 36, p. 9). Water is molded into different structures by the triple helix.     

Solution structure of the second extracellular loop of human thromboxane A2 receptor

Thromboxane A(2) receptor (TP receptor), a prostanoid receptor, belongs to the G protein-coupled receptor family, composed of three intracellular loops and three extracellular loops connecting seven transmembrane helices. The highly conserved extracellular domains of the prostanoid receptors were found in the second extracellular loop (eLP(2)), which was proposed to be involved in ligand recognition. The 3D structure of the eLP(2) would help to further explain the ligand binding mechanism. Analysis of the human TP receptor model generated from molecular modeling based on bacteriorhodopsin crystallographic structure indicated that about 12-14 A separates the N- and C-termini of the extra- and intracellular loops. Synthetic loop peptides whose termini are constrained to this separation are presumably more likely to mimic the native loop structure than the corresponding loop region peptide with unrestricted ends. To test this new concept, a peptide corresponding to the eLP(2) (residues 173-193) of the TP receptor has been made with the N- and C-termini connected by a homocysteine disulfide bond. Through 2D nuclear magnetic resonance (NMR) experiments, complete (1)H NMR assignments, and structural construction, the overall 3D structure of the peptide was determined. The structure shows two beta-turns at residues 180 and 185. The distance between the N- and C-termini of the peptide shown in the NMR structure is 14.2 A, which matched the distance (14.5 A) between the two transmembrane helices connecting the eLP(2) in the TP receptor model. This suggests that the approach using the constrained loop peptides greatly increases the likelihood of solving the whole 3D structures of the extra- and the intracellular domains of the TP receptor. This approach may also be useful in structural studies of the extramembrane loops of other G protein-coupled receptors.     

Intracrine action of angiotensin II in mesangial cells: subcellular distribution of angiotensin II receptor subtypes AT1 and AT2

Biological effects of angiotensin II (AngII) such as regulation of AngII target genes may be triggered by interaction of AngII with intracellular AngII receptor types 1 and 2 (AT₁ and AT₂), defined as intracrine response. The aim of this study was to examine the presence of AT₁ and AT₂ receptors in nuclear membrane of human mesangial cells (HMCs) and evaluate the possible biological effects mediated by intracellular AT₁ through an intracrine mechanism. Subcellular distribution of AT₁ and AT₂ was evaluated by immunofluorescence and by western blot in isolated nuclear extract. Endogenous intracellular synthesis of AngII was stimulated by high glucose (HG). Effects of HG were analyzed in the presence of candesartan, which prevents AngII internalization. Both receptors were found in nuclear membrane. Fluorescein isothiocyanate (FITC)-labeled AngII added to isolated nuclei produced a fluorescence that was reduced in the presence of losartan or PD-123319 and quenched in the presence of both inhibitors simultaneously. HG induced overexpression of fibronectin and increased cell proliferation in the presence of candesartan, indicating an intracrine action of AngII induced by HG. Results showed the presence of nuclear receptors in HMCs that can be activated by AngII through an intracrine response independent of cytoplasmic membrane AngII receptors.

     

Analysis of role of aromatic residues in extracellular loop 2 of Prokineticin receptor 2 in ligand binding probed with genetically encoded photo-crosslinkers

Prokineticin 2 (PK2) and Prokineticin 2 beta (PK2β), products of alternative splicing of pk2 gene, are chemokine-like proteins. While PK2 mediates its biological activities by signaling with the same efficiency through two homologous G protein coupled receptors, prokineticin receptor 1 (PKR1) and prokineticin receptor 2 (PKR2), PK2β is able to bind specifically PKR1.Extracellular loop 2 (ECL2) of chemokine receptors is a part of a transmembrane (TM) ligand binding site. In the ECL2 of PKR2 is present, as well as in all chemokine receptors, an aromatic residue cluster, involving tryptophan 212 localized four residues after an ECL2 conserved cysteine, and Phenylalanine 198 located in the top of TM 4.In this work, the photoactivatable unnatural amino acid p-benzoyl-L-phenylalanine is incorporated by amber codon suppression technology into PKR2 in position 212. Experiments of photoactivatable cross-linking demonstrated the role of tryptophan in position 212 for binding the ligand contacting Tryptophan in position 24. We also analyzed the role of Phenylalanine 198 in the specificity of PKRs binding. The comparison of TM-bundle binding sites between PKR1 and PKR2 revealed that they are completely conserved except for one residue: valine 207 in human PKR1, which is phenylalanine 198 in human PKR2. The F198V mutation in PKR2 permits to obtain a receptor able to bind more efficiently PK2β, a ligand highly specific for PKR1.     

A profile of the residues in the second extracellular loop that are critical for ligand recognition of human prostacyclin receptor

The residues in the second extracellular loop (eLP2) of the prostanoid receptors, which are important for specific ligand recognition, were previously predicted in our earlier studies of the thromboxane A2 receptor (TP) using a combination of NMR spectroscopy and recombinant protein approaches. To further test this hypothesis, another prostanoid receptor, the prostacyclin receptor (IP), which has opposite biological characteristics to that of TP, was used as a model for these studies. A set of recombinant human IPs with site-directed mutations at the nonconserved eLP2 residues were constructed using an Ala-scanning approach, and then expressed in HEK293 and COS-7 cells. The expression levels of the recombinant receptors were six-fold higher in HEK293 cells than in COS-7 cells. The residues important for ligand recognition and binding within the N-terminal segment (G159, Q162, and C165) and the C-terminal segment (L172, R173, M174, and P179) of IP eLP2 were identified by mutagenesis analyses. The molecular mechanisms for the specific ligand recognition of IP were further demonstrated by specific site-directed mutagenesis using different amino acid residues with unique chemical properties for the key residues Q162, L172, R173, and M174. A comparison with the corresponding functional residues identified in TP eLP2 revealed that three (Q162, R173, and M174) of the four residues are nonconserved, and these are proposed to be involved in specific ligand recognition. We discuss the importance of G159 and P179 in ligand recognition through configuration of the loop conformation is discussed. These studies have further indicated that characterization of the residues in the eLP2 regions for all eight prostanoid receptors could be an effective approach for uncovering the molecular mechanisms of the ligand selectivities of the G-protein-coupled receptors.     

Functional trans-inactivation of insulin receptor kinase by growth-inhibitory angiotensin II AT2 receptor

The present study demonstrates negative intracellular cross-talk between angiotensin II type 2 (AT2) and insulin receptors. AT2 receptor stimulation leads to inhibition of insulin-induced extracellular signal-regulated protein kinase (ERK2) activity and cell proliferation in transfected Chinese hamster ovary (CHO-hAT2) cells. We show that AT2 receptor interferes at the initial step of insulin signaling cascade, by impairing tyrosine phosphorylation of the insulin receptor (IR) beta-chain. AT2-mediated inhibition of IR phosphorylation is insensitive to pertussis toxin and is also detected in neuroblastoma N1E-115 and pancreatic acinar AR42J cells that express endogenous receptors. We present evidence that AT2 receptor inhibits the autophosphorylating tyrosine kinase activity of IR, with no significant effect on insulin binding properties. AT2-mediated inactivation of IR does not mainly involve tyrosine dephosphorylation by vanadate-sensitive tyrosine phosphatases nor serine/threonine phosphorylation by protein kinase C. As a consequence of IR inactivation, AT2 receptor inhibits tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1) and signal-regulatory protein (SIRPalpha1) and prevents subsequent association of both IRS-1 and SIRPalpha1 with Src homology 2 (SH2)-containing tyrosine phosphatase SHP-2. Our results thus demonstrate functional trans-inactivation of IR kinase by G protein-coupled AT2 receptor, illustrating a novel mode of negative communication between two families of membrane receptors.     

Evidence of the residues involved in ligand recognition in the second extracellular loop of the prostacyclin receptor characterized by high resolution 2D NMR techniques

In previous studies, we have determined the solution structure of the second extracellular loop (eLP(2)) of the human thromboxane A(2) receptor (TP) and identified the residues in the eLP(2) domain involved in ligand recognition, by using a combination of approaches including a constrained synthetic peptide, 2D NMR spectroscopy, and recombinant proteins. These findings led us to hypothesize that the specific ligand recognition sites may be localized in the eLP(2) for all the prostanoid receptors. To test this hypothesis, we have investigated the ligand recognition site for another prostanoid receptor, the prostacyclin receptor (IP), which mediates an opposite biological function compared to that of the TP receptor. The identification of the interaction between the IP receptor and its agonist, iloprost, was achieved with a constrained synthetic peptide mimicking the eLP(2) region of the receptor. The IP eLP(2) segment was designed and synthesized to form a constrained loop, using a homocysteine disulfide bond connecting the ends of the peptide, based on the distance predicted from the IP receptor model created by homology modeling using the crystal structure of bovine rhodopsin as a template. The evidence of the constrained IP eLP(2) interaction with iloprost was found by the identification of the conformational changes of the eLP(2) induced by iloprost using fluorescence spectroscopy, and was further confirmed by 1D and 2D 1H NMR experiments. In addition, the IP eLP(2)-induced structure of iloprost in solution was elucidated through a complete assignment of the 2D 1H NMR spectra for iloprost in the presence of the IP eLP(2) segment. In contrast, no ordered structure was observed in the 2D 1H NMR experiments for iloprost alone in solution. These studies not only identified that the eLP(2) segment of the IP receptor is involved in ligand recognition, but also solved the 3D solution structure of the bound-form of iloprost, which could be used to study the receptor-ligand interaction in structural terms.     

The first extracellular loop of the Saccharomyces cerevisiae G protein-coupled receptor Ste2p undergoes a conformational change upon ligand binding

In this study of the Saccharomyces cerevisiae G protein-coupled receptor Ste2p, we present data indicating that the first extracellular loop (EL1) of the alpha-factor receptor has tertiary structure that limits solvent accessibility and that its conformation changes in a ligand-dependent manner. The substituted cysteine accessibility method was used to probe the solvent exposure of single cysteine residues engineered to replace residues Tyr(101) through Gln(135) of EL1 in the presence and absence of the tridecapeptide alpha-factor and a receptor antagonist. Surprisingly, many residues, especially those at the N-terminal region, were not solvent-accessible, including residues of the binding-competent yet signal transduction-deficient mutants L102C, N105C, S108C, Y111C, and T114C. In striking contrast, two N-terminal residues, Y101C and Y106C, were readily solvent-accessible, but upon incubation with alpha-factor labeling was reduced, suggesting a pheromone-dependent conformational change limiting solvent accessibility had occurred. Labeling in the presence of the antagonist, which binds Ste2p but does not initiate signal transduction, did not significantly alter reactivity with the Y101C and Y106C receptors, suggesting that the alpha-factor-dependent decrease in solvent accessibility was not because of steric hindrance that prevented the labeling reagent access to these residues. Based on these and previous observations, we propose a model in which the N terminus of EL1 is structured such that parts of the loop are buried in a solvent-inaccessible environment interacting with the extracellular part of the transmembrane domain bundle. This study highlights the essential role of an extracellular loop in activation of a G protein-coupled receptor upon ligand binding.