Additional support for Afrotheria and Paenungulata,the performance of mitochondrial versus nuclear genes,and the impact of data partitions with heterogeneous base composition

We concatenated sequences for four mitochondrial genes (12S rRNA, tRNA valine, 16S rRNA, cytochrome b) and four nuclear genes [aquaporin, alpha 2B adrenergic receptor (A2AB), interphotoreceptor retinoid-binding protein (IRBP), von Willebrand factor (vWF)] into a multigene data set representing 11 eutherian orders (Artiodactyla, Hyracoidea, Insectivora, Lagomorpha, Macroscelidea, Perissodactyla, Primates, Proboscidea, Rodentia, Sirenia, Tubulidentata). Within this data set, we recognized nine mitochondrial partitions (both stems and loops, for each of 12S rRNA, tRNA valine, and 16S rRNA; and first, second, and third codon positions of cytochrome b) and 12 nuclear partitions (first, second, and third codon positions, respectively, of each of the four nuclear genes). Four of the 21 partitions (third positions of cytochrome b, A2AB, IRBP, and vWF) showed significant heterogeneity in base composition across taxa. Phylogenetic analyses (parsimony, minimum evolution, maximum likelihood) based on sequences for all 21 partitions provide 99-100% bootstrap support for Afrotheria and Paenungulata. With the elimination of the four partitions exhibiting heterogeneity in base composition, there is also high bootstrap support (89-100%) for cow + horse. Statistical tests reject Altungulata, Anagalida, and Ungulata. Data set heterogeneity between mitochondrial and nuclear genes is most evident when all partitions are included in the phylogenetic analyses. Mitochondrial-gene trees associate cow with horse, whereas nuclear-gene trees associate cow with hedgehog and these two with horse. However, after eliminating third positions of A2AB, IRBP, and vWF, nuclear data agree with mitochondrial data in supporting cow + horse. Nuclear genes provide stronger support for both Afrotheria and Paenungulata. Removal of third positions of cytochrome b results in improved performance for the mitochondrial genes in recovering these clades.     

A comprehensive structure-function map of the intracellular surface of the human C5a receptor. I. Identification of critical residues

G protein-coupled receptors are one of the largest protein families in nature; however, the mechanisms by which they activate G proteins are still poorly understood. To identify residues on the intracellular face of the human C5a receptor that are involved in G protein activation, we performed a genetic analysis of each of the three intracellular loops and the carboxyl-terminal tail of the receptor. Amino acid substitutions were randomly incorporated into each loop, and functional receptors were identified in yeast. The third intracellular loop contains the largest number of preserved residues (positions resistant to amino acid substitutions), followed by the second loop, the first loop, and lastly the carboxyl terminus. Surprisingly, complete removal of the carboxyl-terminal tail did not impair C5a receptor signaling. When mapped onto a three-dimensional structural model of the inactive state of the C5a receptor, the preserved residues reside on one half of the intracellular surface of the receptor, creating a potential activation face. Together these data provide one of the most comprehensive functional maps of the intracellular surface of any G protein-coupled receptor to date.     

Structure/function analysis of alpha2A-adrenergic receptor interaction with G protein-coupled receptor kinase 2

G protein-coupled receptors (GPCRs) mediate the ability of a diverse array of extracellular stimuli to control intracellular signaling. Many GPCRs are phosphorylated by G protein-coupled receptor kinases (GRKs), a process that mediates agonist-specific desensitization in many cells. Although GRK binding to activated GPCRs results in kinase activation and receptor phosphorylation, relatively little is known about the mechanism of GRK/GPCR interaction or how this interaction results in kinase activation. Here, we used the alpha2A-adrenergic receptor (alpha(2A)AR) as a model to study GRK/receptor interaction because GRK2 phosphorylation of four adjacent serines within the large third intracellular loop of this receptor is known to mediate desensitization. Various domains of the alpha(2A)AR were expressed as glutathione S-transferase fusion proteins and tested for the ability to bind purified GRK2. The second and third intracellular loops of the alpha(2A)AR directly interacted with GRK2, whereas the first intracellular loop and C-terminal domain did not. Truncation mutagenesis identified three discrete regions within the third loop that contributed to GRK2 binding, the membrane proximal N- and C-terminal regions as well as a central region adjacent to the phosphorylation sites. Site-directed mutagenesis revealed a critical role for specific basic residues within these regions in mediating GRK2 interaction with the alpha(2A)AR. Mutation of these residues within the holo-alpha(2A)AR diminished GRK2-promoted phosphorylation of the receptor as well as the ability of the kinase to be activated by receptor binding. These studies provide new insight into the mechanism of interaction and activation of GRK2 by GPCRs and suggest that GRK2 binding is critical not only for receptor phosphorylation but also for full activity of the kinase.     

Intracellular third loops in AT1 and AT2 receptors determine subtype specificity

The recently cloned angiotensin II type 2 (AT2) receptor is a member of the seven transmembrane G-protein coupled receptor superfamily with a relatively low sequence homology with the angiotensin II type 1 (AT1) receptor subtype and counteracts the growth action of AT1 receptor. Intracellular third loops are known to be involved in interactions with various G proteins. We hypothesized that the intracellular third loop plays critical roles in determining the specificity of opposite functions of AT1 and AT2 receptor subtypes and examined this possibility using chimeric AT1 receptor, of which intracellular third loop is replaced with that of AT2 receptor. We transfected this chimeric receptor into PC 12 cells and observed that stimulation of this receptor inhibited extracellular signal-regulated kinase (ERK) activation and induces apoptosis, whereas the binding characteristics of this receptor remained those of ATI receptor. Taken together, these results support the notion that intracellular third loop is the critical determinant for mutually antagonistic AT1 and AT2 receptors' signaling pathways.     

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.     

Molecular Evidence for the Major Clades of Placental Mammals

Higher-level relationships among placental mammals, as well as the historical biogeography of this group against the backdrop of continental fragmentation and reassembly, remain poorly understood. Here, we analyze two independent molecular data sets that represent all placental orders. The first data set includes six genes (A2AB, IRBP, vWF, 12S rRNA, tRNA valine, 16S rRNA; total = 5.71 kb) for 26 placental taxa and two marsupials; the second data set includes 2.95 kb of exon 11 of the BRCA1 gene for 51 placental taxa and four marsupials. We also analyzed a concatenation of these data sets (8.66 kb) for 26 placentals and one marsupial. Unrooted and rooted analyses were performed with parsimony, distance methods, maximum likelihood, and a Bayesian approach. Unrooted analyses provide convincing support for a fundamental separation of placental orders into groups with southern and northern hemispheric origins according to the current fossil record. On rooted trees, one or both of these groups are monophyletic depending on the position of the root. Maximum likelihood and Bayesian analyses with the BRCA1 and combined 8.66 kb data sets provide strong support for the monophyly of the northern hemisphere group (Boreoeutheria). Boreoeutheria is divided into Laurasiatheria (Carnivora + Cetartiodactyla + Chiroptera + Eulipotyphla + Perissodactyla + Pholidota) and Euarchonta (Dermoptera + Primates + Scandentia) + Glires (Lagomorpha + Rodentia). The southern hemisphere group is either monophyletic or paraphyletic, depending on the method of analysis used. Within this group, Afrotheria (Proboscidea + Sirenia + Hyracoidea + Tubulidentata + Macroscelidea + Afrosoricida) is monophyletic. A unique nine base-pair deletion in exon 11 of the BRCA1 gene also supports Afrotheria monophyly. Given molecular dates that suggest that the southern hemisphere group and Boreoeutheria diverged in the Early Cretaceous, a single trans-hemispheric dispersal event may have been of fundamental importance in the early history of crown-group Eutheria. Parallel adaptive radiations have subsequently occurred in the four major groups: Laurasiatheria, Euarchonta + Glires, Afrotheria, and Xenarthra.     

Detection of G protein-activator regions in M4 subtype muscarinic, cholinergic, and alpha 2-adrenergic receptors based upon characteristics in primary structure.

The 14-residue region Arg2410-Lys2423 of the human insulin-like growth factor II receptor possesses the ability to stimulate Gi, the activity being dependent on two structural characteristics: (i) at least two basic residues at the N-terminal side and (ii) the C-terminal motif, B-B-X-B or B-B-X-X-B (where B is a basic residue and X is a non-basic residue). The regions satisfying (i) and (ii) with 10 less than or equal to residue length less than or equal to 26 were located in all of the third inner loops and some of the other intracellular domains of the Gi-coupled M4 sub-type muscarinic cholinergic receptor (M4AChR) and the alpha 2-adrenergic receptor (alpha 2AR). Both the second inner loop 130-147 and the C-terminal portion of the third inner loop 382-400 (MIII) of human M4AChR had the ability to stimulate G proteins with the order Gi approximately Go greater than Gs, but only MIII could activate Gi/Go at nanomolar concentrations. In contrast, the N-terminal portion of the third inner loop 218-228 of human alpha 2AR-C10 activated Gi, Go, and Gs at micromolar concentrations with equal potency, whereas the further C-terminal portion of the third inner loop 301-313 of this receptor lacked the ability to activate any G protein. Among these active regions, only MIII indicated Mg(2+)-dependent Gi-stimulating function. Therefore, the search for the regions satisfying (i) and (ii) was useful to localize the G protein-activating activity of Gi-coupled receptors in limited regions, which were not always in the C-terminal portions of the third intracellular loops and activated G proteins in various modes of actions.     

The third intracellular loop of alpha 2-adrenergic receptors determines subtype specificity of arrestin interaction

Nonvisual arrestins (arrestin-2 and -3) serve as adaptors to link agonist-activated G protein-coupled receptors to the endocytic machinery. Although many G protein-coupled receptors bind arrestins, the molecular determinants involved in binding remain largely unknown. Because arrestins selectively promote the internalization of the alpha(2b)- and alpha(2c)-adrenergic receptors (ARs) while having no effect on the alpha(2a)AR, here we used alpha(2)ARs to identify molecular determinants involved in arrestin binding. Initially, we assessed the ability of purified arrestins to bind glutathione S-transferase fusions containing the third intracellular loops of the alpha(2a)AR, alpha(2b)AR, or alpha(2c)AR. These studies revealed that arrestin-3 directly binds to the alpha(2b)AR and alpha(2c)AR but not the alpha(2a)AR, whereas arrestin-2 only binds to the alpha(2b)AR. Truncation mutagenesis of the alpha(2b)AR identified two arrestin-3 binding domains in the third intracellular loop, one at the N-terminal end (residues 194-214) and the other at the C-terminal end (residues 344-368). Site-directed mutagenesis further revealed a critical role for several basic residues in arrestin-3 binding to the alpha(2b)AR third intracellular loop. Mutation of these residues in the holo-alpha(2b)AR and subsequent expression in HEK 293 cells revealed that the mutations had no effect on the ability of the receptor to activate ERK1/2. However, agonist-promoted internalization of the mutant alpha(2b)AR was significantly attenuated as compared with wild type receptor. These results demonstrate that arrestin-3 binds to two discrete regions within the alpha(2b)AR third intracellular loop and that disruption of arrestin binding selectively abrogates agonist-promoted receptor internalization.     

The first inner loop of endothelin receptor type B is necessary for specific coupling to Galpha 13

Endothelin (EDN) receptor type B (EDNRB) activates serum response factor (SRF) via G(q/11) and G(12/13) G proteins. In this study, we investigated the involvement of intracellular loop sequences of EDNRB in coupling to these G proteins. EDNRB mutants were generated and tested for their abilities to activate SRF in NIH3T3 cells and in the mouse embryonic fibroblast cell line (F(q/11)) lacking both Galpha(q) and Galpha(11). EDNRB can activate SRF in NIH3T3 cells via G(q/11), although it can only activate SRF through G(12/13) in F(q/11) cells. Mutants with mutations in the second and third inner loops of EDNRB functioned in the same manner in both cell lines, either able or unable to activate SRF. This finding suggests that the second and third inner loops of EDNRB either participate or not in coupling to both G(q/11) and G(12/13) but are not specific for either one. However, in the first inner loop, a substitution of three Ala residues for Met(128)-Arg(129)-Asn(130) abolished the ability to activate SRF only in F(q/11) cells, suggesting that this mutation might specifically disrupt the coupling to G(12/13) rather than to G(q/11). Further characterization of this first inner loop mutant revealed that exogenous expression of Galpha(12) or Galpha(q) could restore SRF activation, whereas the expression of Galpha(13) did not. Therefore, we conclude that although the three intracellular loops of EDNRB may be involved in coupling to G proteins, residues Met(128)-Arg(129)-Asn(130) in the first intracellular loop are specifically required for activation of Galpha(13).     

Genetic analysis of the first and third extracellular loops of the C5a receptor reveals an essential WXFG motif in the first loop

The extracellular loops of G protein-coupled receptors (GPCRs) frequently contain binding sites for peptide ligands. However, the mechanism of receptor activation following ligand binding and the influence of the extracellular loops in other aspects of receptor function are poorly understood. Here we report a structure-function analysis of the first and third extracellular loops of the human C5a receptor, a GPCR that binds a 74-amino acid peptide ligand. Amino acid substitutions were randomly incorporated into each loop, and functional receptors were identified in yeast. The first extracellular loop contains a large number of positions that cannot tolerate amino acid substitutions, especially residues within the WXFG motif found in many rhodopsin-like GPCRs, yet disruption of these residues does not alter C5a binding affinity. These results demonstrate an unanticipated role for the first extracellular loop, and the WXFG motif in particular, in ligand-mediated activation of the C5a receptor. This motif likely serves a similar role in other GPCRs. The third extracellular loop, in contrast, contains far fewer preserved residues and appears to play a less essential role in receptor activation.     

Differential determinants for coupling of distinct G proteins with the class B secretin receptor

The secretin receptor is a prototypic class B G protein-coupled receptor that is activated by binding of its natural peptide ligand. The signaling effects of this receptor are mediated by coupling with Gs, which activates cAMP production, and Gq, which activates intracellular calcium mobilization. We have explored the molecular basis for the coupling of each of these G proteins to this receptor using systematic site-directed mutagenesis of key residues within each of the intracellular loop regions, and studying ligand binding and secretin-stimulated cAMP and calcium responses. Mutation of a conserved histidine in the first intracellular loop (H157A and H157R) markedly reduced cell surface expression, resulting in marked reduction in cAMP and elimination of measurable calcium responses. Mutation of an arginine (R153A) in the first intracellular loop reduced calcium, but not cAMP responses. Mutation of a dibasic motif in the second intracellular loop (R231A/K232A) had no significant effects on any measured responses. Mutations in the third intracellular loop involving adjacent lysine and leucine residues (K302A/L303A) or two arginine residues separated by a leucine and an alanine (R318A/R321A) significantly reduced cAMP responses, while the latter also reduced calcium responses. Additive effects were elicited by combining the effective mutations, while combining all the effective mutations resulted in a construct that continued to bind secretin normally, but that elicited no significant cAMP or calcium responses. These data suggest that, while some receptor determinants are clearly shared, there are also distinct determinants for coupling with each of these G proteins.     

The amino terminus of the fourth cytoplasmic loop of rhodopsin modulates rhodopsin-transducin interaction

Rhodopsin is a seven-transmembrane helix receptor that binds and catalytically activates the heterotrimeric G protein transducin (G(t)). This interaction involves the cytoplasmic surface of rhodopsin, which comprises four putative loops and the carboxyl-terminal tail. The fourth loop connects the carboxyl end of transmembrane helix 7 with Cys(322) and Cys(323), which are both modified by membrane-inserted palmitoyl groups. Published data on the roles of the fourth loop in the binding and activation of G(t) are contradictory. Here, we attempt to reconcile these conflicts and define a role for the fourth loop in rhodopsin-G(t) interactions. Fluorescence experiments demonstrated that a synthetic peptide corresponding to the fourth loop of rhodopsin inhibited the activation of G(t) by rhodopsin and interacted directly with the alpha subunit of G(t). A series of rhodopsin mutants was prepared in which portions of the fourth loop were replaced with analogous sequences from the beta(2)-adrenergic receptor or the m1 muscarinic receptor. Chimeric receptors in which residues 310-312 were replaced could not efficiently activate G(t). The defect in G(t) interaction in the fourth loop mutants was not affected by preventing palmitoylation of Cys(322) and Cys(323). We suggest that the amino terminus of the fourth loop interacts directly with G(t), particularly with Galpha(t), and with other regions of the intracellular surface of rhodopsin to support G(t) binding.     

Role of intracellular loops of cannabinoid CB(1) receptor in functional interaction with G(alpha16)

The cannabinoid CB(1) but not the CB(2) receptor was demonstrated to couple via G(alpha16) to activate phospholipase C after co-expression in COS7 cells. Chimeric CB(1)/CB(2) receptors were used as a model to study receptor-G(alpha16) interaction. Sequences of the second and third intracellular loops and the carboxy-terminus were substituted from the CB(1) into the CB(2) receptor. Only the triple mutant with all three regions replaced activated phospholipase C to a similar extent as the CB(1) receptor, suggesting that all three intracellular regions are required for interacting with G(alpha16). Several sub-domains within the third intracellular loop were identified for receptor-G(alpha16) interaction.     

Propylbenzilylcholine mustard labels an acidic residue in transmembrane helix 3 of the muscarinic receptor

Muscarinic acetylcholine receptors were purified from rat forebrain and labeled with [3H]N-(2-chloroethyl)N-(2',3'-[3H2]propyl)-2-aminoethylbenzilate. Cleavage of the labeled muscarinic acetylcholine receptors with a lysine-specific protease yielded labeled, glycosylated peptides about 130 and 200 residues in length, which came from different receptor sequences. The probable cleavage sites are in the second intracellular loop and in the second extracellular or third intracellular loop. The N-terminal 130 residues are disulfide-bonded to another part of the receptor structure, supporting the presence of a link between the second and third extracellular loops. The [3H]propylbenzilylcholine mustard-receptor link is cleaved by nucleophiles, acids, and bases under denaturing conditions, suggesting modification of an acidic residue. Cyanogen bromide cleavage points to transmembrane helix 3 as the site of label attachment.     

Mutational analysis of predicted intracellular loop domains of human motilin receptor

Motilin is an important endogenous regulator of gastrointestinal motor function, mediated by the class I G protein-coupled motilin receptor. Motilin and erythromycin, two chemically distinct full agonists of the motilin receptor, are known to bind to distinct regions of this receptor, based on previous systematic mutagenesis of extracellular regions that dissociated the effects on these two agents. In the present work, we examined the predicted intracellular loop regions of this receptor for effects on motilin- and erythromycin-stimulated activity. We prepared motilin receptor constructs that included sequential deletions throughout the predicted first, second, and third intracellular loops, as well as replacing the residues in key regions with alanine, phenylalanine, or histidine. Each construct was transiently expressed in COS cells and characterized for motilin- and erythromycin-stimulated intracellular calcium responses and for motilin binding. Deletions of receptor residues 63-66, 135-137, and 296-301 each resulted in substantial loss of intracellular calcium responses to stimulation by both motilin and erythromycin. Constructs with mutations of residues Tyr66, Arg136, and Val299 were responsible for the negative impact on biological activity stimulated by both agonists. These data suggest that action by different chemical classes of agonists that are known to interact with distinct regions of the motilin receptor likely yield a common activation state of the cytosolic face of this receptor that is responsible for interaction with its G protein. The identification of functionally important residues in the predicted cytosolic face provides strong candidates for playing roles in receptor-G protein interaction.     

Functional analysis of the cytoplasmic domains of the human thyrotropin receptor by site-directed mutagenesis

The thyrotropin (TSH) receptor belongs to a family of guanine nucleotide protein-coupled receptors with seven transmembrane-spanning regions joined regulatory together by extracellular and intracellular loops. The cytoplasmic domain comprises three cytoplasmic loops and a cytoplasmic tail that are likely to be important in coupling of the receptor to the guanine nucleotide proteins. To address the question of which portions of the cytoplasmic domain of the TSH receptor are important in this process, we have altered groups of amino acids in the region of the TSH receptor by site-directed mutagenesis. Because of the low affinity of TSH binding to the TSH receptor mutated in the amino terminus of the second cytoplasmic loop and the amino terminus of the cytoplasmic tail, definitive conclusions cannot be made regarding the roles of these regions in signal transduction. However, our data indicate that the first cytoplasmic loop (residues 441-450), the carboxyl-terminal region of the second cytoplasmic loop (residues 528-537), and the carboxyl-terminal (but not the amino-terminal) region of the third cytoplasmic loop (residues 617-625) are important in the ability of the TSH receptor to mediate an increase in intracellular cAMP production. Furthermore, two-thirds of the carboxyl-terminal end of the cytoplasmic tail (residues 709-764; corresponding to the region not conserved between the TSH and lutropin/chorionic gonadotropin receptors) can be removed without functional impairment of the TSH receptor.     

The role of conserved extracellular cysteine residues in vasopressin V2 receptor function and properties of two naturally occurring mutant receptors with additional extracellular cysteine residues

The G protein-coupled vasopressin V2 receptor (V2 receptor) contains a pair of conserved cysteine residues (C112 and C192) which are thought to form a disulfide bond between the first and second extracellular loops. The conserved cysteine residues were found to be important for the correct formation of the ligand binding domain of some G protein-coupled receptors. Here we have assessed the properties of the V2 receptor after site-directed mutagenesis of its conserved cysteine residues in transiently transfected human embryonic kidney (HEK 293) cells. Mutant receptors (C112S, C112A and C192S, C192A) were non-functional and located mostly in the cell's interior. The conserved cysteine residues of the V2 receptor are thus not only important for the structure of the ligand binding domain but also for efficient intracellular receptor transport. In addition to the functional significance of the conserved cysteine residues, we have also analyzed the defects of two mutant V2 receptors which cause X-linked nephrogenic diabetes insipidus (NDI) by the introduction of additional cysteine residues into the second extracellular loop (mutants G185C, R202C). These mutations are assumed to impair normal disulfide bond formation. Mutant receptor G185C and R202C were efficiently transported to the plasma membrane but were defective in ligand binding. Only in the case of the mutant receptor R202C, the more sensitive adenylyl cyclase activity assay revealed vasopressin-stimulated cAMP formation with a 35-fold increased EC(50) value and with a reduced EC(max), indicating that ligand binding is not completely abolished. Taking the unaffected intracellular transport of both NDI-causing mutant receptors into account, our results indicate that the observed impairment of ligand binding by the additional cysteine residues is not due to the prevention of disulfide bond formation between the conserved cysteine residues.     

Mutating the four extracellular cysteines in the chemokine receptor CCR6 reveals their differing roles in receptor trafficking,ligand binding,and signaling

CCR6 is the receptor for the chemokine MIP-3 alpha/CCL20. Almost all chemokine receptors contain cysteine residues in the N-terminal domain and in the first, second, and third extracellular loops. In this report, we have studied the importance of all cysteine residues in the CCR6 sequence using site-directed mutagenesis and biochemical techniques. Like all G protein-coupled receptors, mutating disulfide bond-forming cysteines in the first (Cys118) and second (Cys197) extracellular loops in CCR6 led to complete elimination of receptor activity, which for CCR6 was also associated with the accumulation of the receptor intracellularly. Although two additional cysteines in the N-terminal region and the third extracellular loop, which are present in almost all chemokine receptors, are presumed to form a disulfide bond, this has not been demonstrated experimentally for any of these receptors. We found that mutating the cysteines in the N-terminal domain (Cys36) and the third extracellular loop (Cys288) neither significantly affected receptor surface expression nor completely abolished receptor function. Importantly, contrary to several previous reports, we demonstrated directly that instead of forming a disulfide bond, the N-terminal cysteine (Cys36) and the third extracellular loop cysteine (Cys288) contain free SH groups. The cysteine residues (Cys36 and Cys288), rather than forming a disulfide bond, may be important per se. We propose that CCR6 forms only a disulfide bond between the first (Cys118) and second (Cys197) extracellular loops, which confines a helical bundle together with the N-terminus adjacent to the third extracellular loop, creating the structural organization critical for ligand binding and therefore for receptor signaling.     

Assembling NMR structures for the intracellular loops of the human thromboxane A2 receptor: implication of the G protein-coupling pocket

It has been reported that the multiple intracellular loops (iLPs) of the thromboxane A₂ receptor (TP) are involved in the receptor G protein coupling. In this study, a high-resolution 2D NMR technique was used to determine the 3D structures of the first, second, and third iLPs of the TP using synthetic peptides constrained into the loop structures. 2D ¹H NMR spectra, TOCSY and NOESY were obtained for the two peptides from proton NMR experiments. The NMR data was processed and assigned through the Felix 2000 program. Standard methods were used to acquire sequence-specific assignments. Structure calculations were processed through DGII and NMR refinement programs within the Insight II program. We were able to calculate and use the NOE constraints to obtain the superimposed structure of 10 structures for each iLP peptide. The NMR-determined structures of the iLP peptides were used to refine a homology model of the TP. A 3D G-protein-binding cavity, formed by the three intracellular loops, was predicted by the docking of the C-terminal domain of the Gαq. Based on the structural model and the previous mutagenesis studies, the residues, R130, R60, C223, F138, L360, V361, E358 and Y359, which are important for interaction with the G protein, were further highlighted. These results reveal the possibly important molecular mechanisms in TP signaling and provide structural information to characterize other prostanoid receptor signalings.     

Theoretical study of the electrostatically driven step of receptor-G protein recognition

This study proposes a theoretical model describing the electrostatically driven step of the alpha 1 b-adrenergic receptor (AR)-G protein recognition. The comparative analysis of the structural-dynamics features of functionally different receptor forms, i.e., the wild type (ground state) and its constitutively active mutants D142A and A293E, was instrumental to gain insight on the receptor-G protein electrostatic and steric complementarity. Rigid body docking simulations between the different forms of the alpha 1 b-AR and the heterotrimeric G alpha q, G alpha s, G alpha i1, and G alpha t suggest that the cytosolic crevice shared by the active receptor and including the second and the third intracellular loops as well as the cytosolic extension of helices 5 and 6, represents the receptor surface with docking complementarity with the G protein. On the other hand, the G protein solvent-exposed portions that recognize the intracellular loops of the activated receptors are the N-terminal portion of alpha 3, alpha G, the alpha G/alpha 4 loop, alpha 4, the alpha 4/beta 6 loop, alpha 5, and the C-terminus. Docking simulations suggest that the two constitutively active mutants D142A and A293E recognize different G proteins with similar selectivity orders, i.e., G alpha q approximately equal to G alpha s > G alpha i > G alpha t. The theoretical models herein proposed might provide useful suggestions for new experiments aiming at exploring the receptor-G protein interface.