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M Cottet,
L Albizu,
S Perkovska,
F Jean-Alphonse,
R Rahmeh,
H Orcel,
C Méjean,
S Granier,
C Mendre, B Mouillac,
T Durroux
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ABSTRACT: The G protein-coupled receptors (GPCRs) play a major role in the regulation of physiological function. The emergence of the concept of GPCR oligomerization deeply modifies our understanding of their functioning. Much more than a simple association leading to an independent functioning, the GPCR oligomerization affects various steps such as membrane targeting of the receptors, binding of ligands, coupling to the intracellular pathways and internalization. Although significant advances have been performed in proving the existence of GPCR oligomers, its physiological impact remains to be established. Vasopressin and oxytocin receptors have constituted interesting experimental models in oligomer analysis. Because of the pharmacological tools available regarding these receptors and their expression at a high level in various tissues they can constitute very promising models to study the consequences of oligomerization in physiology.
Current Opinion in Pharmacology 11/2009; 10(1):59-66. · 6.86 Impact Factor
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ABSTRACT: In annelids, it has been established that arginine-vasopressin (AVP)/oxytocin (OT) superfamily peptides are involved in the maintenance of water and electrolyte homeostasis as well as reproduction. At present, there is little information on their receptors. In this study, we report the characterization of a 1.7 kb cDNA for an AVP-related receptor from the leech Theromyzon tessulatum. The open reading frame encodes a 435-aminoacid transmembrane protein that displays seven segments of hydrophobic amino acids, typical of G-protein-coupled receptors. The overall predicted protein exhibits about 30% amino-acid identities to other invertebrate, as well as vertebrate, AVP/OT receptor family members, and displays conserved characteristic features belonging to the AVP/OT receptor superfamily. RT-PCR expression experiments showed that mRNA is expressed in the genital tract, the ovary and the brain. The receptor expression is stage specific, showing a weak expression after the two first blood meals, increasing dramatically after the last blood meal during the period of sexual maturation and disappearing after egg laying. Thus, the leech AVP-related receptor may mediate reproductive functions. When expressed in COS-7 cells, the receptor binds ligands with the following rank order of potency: AVP= Arg-vasotocin >Arg-conopressin >mesotocin = OT = Lys-conopressin=isotocin>annetocin. This shows an AVP-like pharmacological profile. The transfected receptor mediates AVP-induced accumulation of inositol phosphates, indicating that the leech AVP-related receptor is functional. This study describes the characterization of a novel AVP/OT superfamily receptor in annelids, which are considered the most distant group of coelomate metazoans possessing a functional AVP/OT-related endocrine system.
Journal of Endocrinology 01/2005; 184(1):277-89. · 3.55 Impact Factor
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ABSTRACT: Using a segment strategy, we have synthesized four iodinated photoactivatable cyclic peptidic ligands of oxytocin, bearing a beta-mercapto-betabeta-cyclopentamethylene propionic group (Pmp) on their N-terminus. All the syntheses were RP-HPLC monitored, and the compounds were HPLC purified. They were characterized by 1H NMR, MALDI-TOF, or FAB mass spectrometries. The affinities of Pmp-Tyr(Me)-Ile-Thr-Asn-Cys-Gly-Orn-Phe(3I,4N3)-NH2 (20), Pmp-Tyr-Ile-Thr-Asn-Cys-Gly-Orn-Phe(3I,4N3)-NH2 (21), Pmp-Tyr(Me)-Ile-Thr-Asn-Cys-Pro-Orn-Phe(3I,4N3)-NH2 (22), and Pmp-Tyr-Ile-Thr-Asn-Cys-Pro-Orn-Phe(3I,4N3)-NH2 (23) were evaluated as inhibition constants (K(i), in nM) for the human oxytocin receptor expressed in Chinese hamster ovary cells by displacement of a radioiodinated disulfide-cyclized antagonist (Elands et al. Eur. J. Pharmacol. 1987, 147, 197-207). The most potent of them, compound 22, was synthesized by another method in order to allow its radiolabeling by 125I. Its dissociation constant (K(d)) for the human oxytocin receptor, directly measured in saturation studies, was 0.25 +/- 0.04 nM, and its antagonist properties were determined by inactivation of phospholipase C, thus obtaining an inactivation constant (K(inact)) of 0.18 +/- 0.02 nM, evaluated by inositol phosphate accumulation. This compound is a very good tool for the mapping of peptidic antagonist binding sites in the human oxytocin receptor.
Journal of Medicinal Chemistry 09/2001; 44(18):3022-30. · 5.25 Impact Factor
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C Breton,
H Chellil,
M Kabbaj-Benmansour,
E Carnazzi,
R Seyer,
S Phalipou,
D Morin,
T Durroux,
H Zingg,
C Barberis, B Mouillac
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ABSTRACT: Understanding of the molecular determinants responsible for antagonist binding to the oxytocin receptor should provide important insights that facilitate rational design of potential therapeutic agents for the treatment of preterm labor. To study ligand/receptor interactions, we used a novel photosensitive radioiodinated antagonist of the human oxytocin receptor, d(CH(2))(5) [Tyr(Me)(2),Thr(4),Orn(8),Phe(3(125)I,4N(3))-NH(2)9]vasotocin. This ligand had an equivalent high affinity for human oxytocin and V(1a) vasopressin receptors expressed in Chinese hamster ovary cells. Taking advantage of this dual specificity, we conducted photoaffinity labeling experiments on both receptors. Photolabeled oxytocin and V(1a) receptors appeared as a unique protein band at 70-75 kDa and two labeled protein bands at 85-90 and 46 kDa, respectively. To identify contact sites between the antagonist and the receptors, the labeled 70-75- and the 46-kDa proteins were cleaved with CNBr and digested with Lys-C and Arg-C endoproteinases. The fragmentation patterns allowed the identification of a covalently labeled region in the oxytocin receptor transmembrane domain III consisting of the residues Leu(114)-Val(115)-Lys(116). Analysis of contact sites in the V(1a) receptor led to the identification of the homologous region consisting of the residues Val(126)-Val(127)-Lys(128). Binding domains were confirmed by mutation of several CNBr cleavage sites in the oxytocin receptor and of one Lys-C cleavage site in the V(1a) receptor. The results are in agreement with previous experimental data and three-dimensional models of agonist and antagonist binding to members of the oxytocin/vasopressin receptor family.
Journal of Biological Chemistry 08/2001; 276(29):26931-41. · 4.77 Impact Factor
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N Cotte,
M N Balestre,
A Aumelas,
E Mahé,
S Phalipou,
D Morin,
M Hibert,
M Manning,
T Durroux,
C Barberis, B Mouillac
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ABSTRACT: Despite their opposite effects on signal transduction, the nonapeptide hormone arginine-vasopressin (AVP) and its V1a receptor-selective cyclic peptide antagonist d(CH2)5[Tyr(Me)2]AVP display homologous primary structures, differing only at residues 1 and 2. These structural similarities led us to hypothesize that both ligands could interact with the same binding pocket in the V1a receptor. To determine receptor residues responsible for discriminating binding of agonist and antagonist ligands, we performed site-directed mutagenesis of conserved aromatic and hydrophilic residues as well as nonconserved residues, all located in the transmembrane binding pocket of the V1a receptor. Mutation of aromatic residues of transmembrane region VI (W304, F307, F308) reduced affinity for the d(CH2)5[Tyr(Me)2]AVP and markedly decreased affinity for the unrelated strongly hydrophobic V1a-selective nonpeptide antagonist SR 49059. Replacement of these aromatic residues had no effect on AVP binding, but increased AVP-induced coupling efficacy of the receptor for its G protein. Mutating hydrophilic residues Q108, K128 and Q185 in transmembrane regions II, III and IV, respectively, led to a decrease in affinity for both agonists and antagonists. Finally, the nonconserved residues T333 and A334 in transmembrane region VII, controlled the V1a/V2 binding selectivity for both nonpeptide and cyclic peptide antagonists. Thus, because conserved aromatic residues of the V1a receptor binding pocket seem essential for antagonists and do not contribute at all to the binding of agonists, we propose that these residues differentiate agonist vs. antagonist ligand binding.
European Journal of Biochemistry 08/2000; 267(13):4253-63. · 3.58 Impact Factor
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ABSTRACT: A novel photoactivatable linear peptide antagonist selective for the V(1a) vasopressin receptor, [(125)I][Lys(3N(3) Phpa)(8)]HO-LVA, was synthesized, characterized, and used to photolabel the human receptor expressed in Chinese hamster ovary cells. Two specific glycosylated protein species at 85-90 and 46 kDa were covalently labeled, a result identical to that obtained with a previous photosensitive ligand, [(125)I]3N(3)Phpa-LVA (Phalipou, S., Cotte, N. , Carnazzi, E., Seyer, R., Mahe, E., Jard, S., Barberis, C., and Mouillac, B. (1997) J. Biol. Chem. 272, 26536-26544). To identify contact sites between the new photoreactive analogue and the V(1a) receptor, the labeled receptors were digested with Lys-C or Asp-N endoproteinases and chemically cleaved with CNBr. Fragmentation with CNBr, Lyc-C, and Asp-N used alone or in combination, led to the identification of a restricted receptor region spanning the first extracellular loop. The results established that sequence Asp(112)-Pro(120) could be considered as the smallest covalently labeled fragment with [(125)I][Lys(3N(3)Phpa)(8)]HO-LVA. Based on the present experimental result and on previous photoaffinity labeling data obtained with [(125)I]3N(3)Phpa-LVA (covalent attachment to transmembrane domain VII), three-dimensional models of the antagonist-bound receptors were constructed and then verified by site-directed mutagenesis studies. Strikingly, these two linear peptide antagonists, when bound to the V(1a) receptor, could adopt a pseudocyclic conformation similar to that of the cyclic agonists. Despite divergent functional properties, these peptide antagonists could interact with a transmembrane-binding site significantly overlapping that of the natural hormone vasopressin.
Journal of Biological Chemistry 09/1999; 274(33):23316-27. · 4.77 Impact Factor
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ABSTRACT: The substitution, in the human V2 vasopressin receptor, of the aspartate at position 136 by alanine leads to agonist-independent activation of this mutant V2 receptor. Pharmacological studies of the D136A V2 receptor helped us in characterizing different V2 receptor antagonists. SR-121463A and OPC-31260, two non-peptide antagonists, behaved as inverse agonists, while two cyclic peptides d(CH2)5[D-Tyr(Et)2,-Val4,Tyr-NH(2)9]AVP and d(CH2)5[D-Ile2,Ile4,Tyr-NH(2)9]AVP known to be V2 antagonists, demonstrated clear partial agonist properties. The finding of a constitutively activated human V2 receptor represents a useful tool in characterizing V2 receptor antagonist ligands.
FEBS Letters 01/1999; 441(3):470-5. · 3.54 Impact Factor
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Y Ala,
D Morin, B Mouillac,
N Sabatier,
R Vargas,
N Cotte,
M Déchaux,
C Antignac,
M F Arthus,
M Lonergan,
M S Turner,
M N Balestre,
G Alonso,
M Hibert,
C Barberis,
G N Hendy,
D G Bichet,
S Jard
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ABSTRACT: X-linked nephrogenic diabetes insipidus (NDI) is a rare disease with defective renal and extrarenal arginine vasopressin V2 receptor responses due to mutations in the AVPR2 gene in Xq28. To study the cause of loss of function of mutant V2 receptors, we expressed 12 mutations (N55H, L59P, L83Q, V88M, 497CC-->GG, deltaR202, I209F, 700delC, 908insT, A294P, P322H, P322S) in COS-7 cells. Eleven of these, including P322H, were characterized by a complete loss of function, but the mutation P322S demonstrated a mild clinical and in vitro phenotype. This was characterized by a late diagnosis without any growth or developmental delay and a significant increase in urine osmolality after intravenous 1-deamino[D-Arg8]AVP administration. In vitro, the P322S mutant was able to partially activate the Gs/adenylyl cyclase system in contrast to the other V2R mutants including P322H, which were completely inactive in this regard. This showed not only that Pro 322 is important for proper V2R coupling, but also that the degree of impairment is strongly dependent on the identity of the substituting amino acid. Three-dimensional modeling of the P322H and P322S mutant receptors suggested that the complete loss of function of the P322H receptor could be due, in part, to hydrogen bond formation between the His 322 side chain and the carboxyl group of Asp 85, which does not occur in the P322S receptor.
Journal of the American Society of Nephrology 10/1998; 9(10):1861-72. · 9.66 Impact Factor
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ABSTRACT: The steroid hormone progesterone (P4) is essential for establishing and maintaining pregnancy in mammals. One of its functions includes maintenance of uterine quiescence by decreasing uterine sensitivity to the uterotonic peptide hormone oxytocin. Although it is generally held that steroid hormones such as P4 act at a genomic level by binding to nuclear receptors and modulating the expression of specific target genes, we show here that the effect of P4 on uterine sensitivity to oxytocin involves direct, non-genomic action of P4 on the uterine oxytocin receptor (OTR), a member of the G-protein-coupled receptor family. P4 inhibits oxytocin binding to OTR-containing membranes in vitro, binds with high affinity to recombinant rat OTR expressed in CHO cells, and suppresses oxytocin-induced inositol phosphate production and calcium mobilization. These effects are highly steroid- and receptor-specific, because binding and signalling functions of the closely related human OTR are not affected by P4 itself but by the P4 metabolite 5beta-dihydroprogesterone. Our findings provide the first evidence for a direct interaction between a steroid hormone and a G-protein-coupled receptor and define a new level of crosstalk between the peptide- and steroid-hormone signalling pathways.
Nature 04/1998; 392(6675):509-12. · 36.28 Impact Factor
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Journal of Endocrinology 03/1998; 156(2):223-9. · 3.55 Impact Factor
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Advances in experimental medicine and biology 02/1998; 449:359-61. · 1.09 Impact Factor
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ABSTRACT: Our recent studies have shown that regulation of uterine oxytocin (OT) binding involves at least two different mechanism: Estradiol (E2)-induced upregulation is accompanied by an increase in OT receptor (OTR) mRNA accumulation, implying that the E2 effect is mediated via increased OTR gene transcription and/or OTR mRNA stabilization. In contrast, P (P)-induced OTR down-regulation occurs via a novel non-genomic mechanism, involving a direct interaction of P with the OTR at the level of the cell membrane. We found that P specifically binds to the OTR and inhibits its ligand binding and signalling functions. Physiological levels of P repress in vitro the ligand binding capacity (Bmax) of the OTR by > 50%. When expressed in CHO cells, the OTR provides a high affinity (Kd: 20nM) membrane binding site for P. OT-induced inositol phosphate production and intracellular calcium mobilization is inhibited 85% and 90%, respectively, by P. These effects are specific as signalling and binding functions of the closely related V1a vasopressin receptor remain unaffected by P, and as other, related steroids are devoid of any effect on OTR binding or signalling functions. The present observation of a specific interaction of a steroid with a G-protein-linked receptor defines a new mechanism of non-genomic steroid action and uncovers a novel level of crosstalk between steroid and peptide hormone action.
Advances in experimental medicine and biology 02/1998; 449:287-95. · 1.09 Impact Factor
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ABSTRACT: The study of antagonist-binding domains of the human V1a vasopressin receptor was performed using a radioiodinated photoreactive peptide antagonist. This ligand displayed a high affinity for the receptor expressed in Chinese hamster ovary cell membranes, and specifically labeled two protein bands with apparent molecular mass at 85-90 and 46 kDa. Our results clearly show that the V1a receptor is degraded during incubation with the ligand and that the 46-kDa species is probably the result of the 85-90-kDa species proteolytic cleavage. Truncation of the receptor was then confirmed by deglycosylation with N-glycosidase F. A monoclonal antibody directed against a c-Myc epitope added at the receptor NH2 terminus allowed immunoprecipitation of the 85-90-kDa photolabeled species. The 46-kDa photolabeled protein never immunoprecipitated, indicating that the truncated form of the receptor lacks the NH2 terminus region. To localize photolabeled domains of the receptor, the 46-kDa protein was cleaved with V8 and/or Lys-C endoproteinases. The identity of the smallest photolabeled fragment, observed at approximately 6 kDa, was then confirmed by mutation of the potential V8 cleavage sites. Our results indicate that covalent labeling of the vasopressin V1a receptor with the photoreactive antagonist occurs in a region including transmembrane domain VII (residues Asn327-Lys370).
Journal of Biological Chemistry 11/1997; 272(42):26536-44. · 4.77 Impact Factor
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ABSTRACT: We have synthesized and fully characterized by fast-atom-bombardment-mass, NMR and ultraviolet spectroscopies the vasopressin antagonist 3-azidophenylpropionyl-D-Tyr(Me)-Phe-Gln-Asn-Arg-Pro-Arg-Tyr(3I )-NH2. Easily radioiodinatable just before use, it has a high affinity for the natural rat liver V1a receptor [dissociation constant (Kd) = 54 +/- 20 pM; Carnazzi, E., Aumelas, A., Barberis, C., Guillon, G. & Seyer, R. (1994) J. Med. Chem. 37, 1841-1849] and for both the rat vasopressin V1a receptor expressed in Spodoptera frugiperda 9 cells (Sf9 cells, Kd = 688 +/- 35 pM) and in COS-7 cells (Kd = 320 +/- 20 pM). This probe labels specifically the V1a receptors in an ultraviolet-dependent manner, and binds covalently to about 12% of the receptors with high stability over several days, even in dissociation or solubilization conditions. SDS/PAGE studies and autoradiographic analyses of the photolabeled receptors reveal a single band (49.5 kDa) and two bands (63 kDa and 93.6 kDa) for receptor-probe associations obtained in Sf9 and COS-7 cells respectively. These molecular masses are consistent with non-glycosylated and highly glycosylated forms of the receptor, according to each expression system. In rat liver membranes, we have identified apparent molecular masses of about 32, 45 and more than 67 kDa. We finally demonstrated a proteolysis of the receptor that appeared to be Zn2+ and leupeptin sensitive. The high potency of this ligand is promising for the monitoring of the purification of the V1a receptor and for mapping its antagonist-binding site.
European Journal of Biochemistry 08/1997; 247(3):906-13. · 3.58 Impact Factor
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Y Ala,
D Morin,
E Mahé,
N Cotte, B Mouillac,
S Jard,
C Barberis,
E Tribollet,
J J Dreifuss,
W H Sawyer,
N C Wo,
W Y Chan,
A S Kolodziejczyk,
L L Cheng,
M Manning
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ABSTRACT: A vasopressin receptor antagonist, [1-(beta-mercapto-beta,beta-pentamethylenepropionic acid), 2-o-ethyl-D-tyrosine, 4-valine, 9-tyrosylamide] arginine vasopressin (d(CH2)5[o-ethyl-D-Tyr2,Val4,Tyr-NH9(2)]AVP), has been prepared. This antagonist is a potent antiantidiuretic, antivasopressor and antioxytocic peptide with pA2 values of 7.69-7.94 and affinities of 1.12-11.0 nM. When radioiodinated at the phenyl moiety of the tyrosylamide residue at position 9, this peptide was demonstrated to bind to vasopressin V2 and V1a receptors with a dissociation constant of 0.22-0.75 nM. This ligand is a good tool for further studies on human vasopressin V2 receptor localization and characterization, when used in combination with a selective vasopressin V1a ligand.
European Journal of Pharmacology 08/1997; 331(2-3):285-93. · 2.52 Impact Factor
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ABSTRACT: Sustained stimulation of the beta2-adrenoceptor leads to a desensitization of the receptor-mediated adenylyl cyclase stimulation. While desensitization promoted by nanomolar concentrations of isoproterenol involves the phosphorylation of the beta2-adrenoceptor by protein kinase A alone, both protein kinase A- and beta-adrenoceptor kinase-mediated phosphorylation leading to the binding of beta-arrestin contribute to the desensitization evoked by micromolar concentrations of agonist. In the present study, we assessed the influence of receptor density on the patterns of desensitization induced by these two different levels of stimulation. Murine L cells were transfected with a cDNA encoding the human beta2-adrenoceptor and clonal cell lines expressing various levels of beta2-adrenoceptor were used for the study. In cell lines expressing the highest number of receptor, approx. 150000 sites/cell (approx. 3000 fmol/mg of membrane proteins), pretreatment with micromolar concentrations of isoproterenol causes a desensitization pattern characterized by a reduction in both the potency and the efficacy of isoproterenol to further stimulate the adenylyl cyclase activity. In contrast, desensitization induced by 10 nM isoproterenol resulted only in a decrease in the potency of isoproterenol. This distinct pattern of desensitization is not seen in cells expressing 12000 receptors/cell (approx. 200 fmol/mg of membrane proteins) and, in that case, pretreatment with 10 nM isoproterenol leads to a reduction in both the sensitivity and the maximal response. Similar effects on the beta-adrenoceptor-stimulated adenylyl cyclase were observed in these cells following treatment with dibutyryl cAMP. Receptor density therefore dramatically influences the pattern of desensitization evoked by low level of stimulation. The results also demonstrate that although different molecular events are involved in the desensitization evoked by different levels of stimulation, its phenotypic expression can be qualitatively identical in cells expressing a relatively small number of receptors. Hence, protein kinase A-mediated desensitization cannot be qualitatively distinguished from the beta-adrenoceptor kinase-mediated process in these cells.
European Journal of Pharmacology 06/1997; 326(1):75-84. · 2.52 Impact Factor
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B Chini, B Mouillac,
Y Ala,
M N Balestre,
S Trumpp-Kallmeyer,
J Hoflack,
J Elands,
M Hibert,
M Manning,
S Jard,
C Barberis
Annals of the New York Academy of Sciences 06/1997; 812:218-21. · 3.15 Impact Factor
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ABSTRACT: We investigated the mechanisms that regulate the efficacy of agonists in the arginine-vasopressin (AVP)/oxytocin (OT) receptor system. In this paper, we present evidence that AVP, a full agonist of the vasopressin receptors, acts as a partial agonist on the oxytocin receptor. We also found that AVP becomes a full agonist when two aromatic residues of the oxytocin receptor are replaced by the residues present at equivalent positions in the vasopressin receptor subtypes. Our results indicate that these two residues modulate the response of the oxytocin receptor to the partial agonist AVP.
FEBS Letters 12/1996; 397(2-3):201-6. · 3.54 Impact Factor
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ABSTRACT: We previously showed that substitution of a glycine residue for the palmitoylated cysteine 341 of the human beta2-adrenergic receptor (Gly341beta2AR), increases the basal level of the receptor phosphorylation and reduces its ability to functionally interact with Gs. In the present study, we show that additional mutation of serines 345 and 346 (Ala345,346Gly341beta2AR) restored normal phosphorylation and receptor-Gs coupling, thus suggesting that the increased phosphorylation of this site, rather than the lack of palmitoylation per se, is responsible for the poor coupling of the unpalmitoylated receptor. This is supported by the observation that chemical depalmitoylation of purified beta2AR did not affect the ability of the receptor to stimulate adenylyl cyclase in reconstitution assays. Furthermore, mutation of Ser345,346 in a wild type receptor background (Ala345,346beta2AR) significantly decreased the rate of agonist-promoted desensitization of the receptor-stimulated adenylyl cyclase activity, supporting a role for this phosphorylation site in regulating the functional coupling of the receptor. Since serines 345 and 346 are located in a putative cyclic AMP-dependent protein kinase (PKA) phosphorylation site immediately downstream of the palmitoylated cysteine 341, the hypothesis that the accessibility of this site may be regulated by the receptor palmitoylation state was further assessed in vitro. In membrane phosphorylation assays, Gly341beta2AR was found to be a better substrate for PKA than the wild type receptor, thus supporting the notion that palmitoylation restrains access of the phosphorylation site to the enzyme. Taken together, the data demonstrate that palmitoylation of cysteine 341 controls the phosphorylation state of the PKA site located in the carboxyl tail of the beta2AR and by doing so modulates the responsiveness of the receptor.
Journal of Biological Chemistry 09/1996; 271(35):21490-7. · 4.77 Impact Factor
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ABSTRACT: To identify receptor functional domains underlying binding of the neurohypophysial hormones vasopressin (AVP) and oxytocin (OT), we have constructed a three-dimensional (3D) model of the V1a vasopressin receptor subtype and docked the endogenous ligand AVP. To verify and to refine the 3D model, residues likely to be involved in agonist binding were selected for site-directed mutagenesis. Our experimental results suggest that AVP, which is characterized by a cyclic structure, could be completely buried into a 15-20-A deep cleft defined by the transmembrane helices of the receptor and interact with amino acids located within this region. Moreover, the AVP-binding site is situated in a position equivalent to that described for the cationic neurotransmitters. Since all mutated residues are highly conserved in AVP and OT receptors, we propose that the same agonist-binding site is shared by all members of this receptor family. In contrast, the affinity for the antagonists tested, including those with a structure closely related to AVP, is not affected by mutations. This indicates a different binding mode for agonists and antagonists in the vasopressin receptor.
Journal of Biological Chemistry 11/1995; 270(43):25771-7. · 4.77 Impact Factor
