The role of N-terminal and C-terminal Arg residues from BK on interaction with kinin B2 receptor.

Bradykinin (BK) is a nonapeptide important for several physiological processes such as vasodilatation, increase in vascular permeability and release of inflammatory mediators. BK performs its actions by coupling to and activating the B2 receptor, a family A G-protein coupled receptor. Using a strategy which allows systematical monitoring of BK R1 and R9 residues and B2 receptor acidic residues Glu5.35(226) and Asp6.58(298), our study aims at clarifying the BK interaction profile with the B2 receptor [receptor residue numbers are normalized according to Ballesteros and Weinstein, Methods Neurosci. 25 (1995), pp. 366-428) followed by receptor sequence numbering in brackets]. N- and C-terminal analogs of BK (-A1, -G1, -K1, -E1 and BK-A9) were tested against wild type B2, Glu5.35(226)Ala and Asp6.58(298)Ala B2 mutant receptors for their affinity and capability to elicit responses by mechanical recordings of isolated mice stomach fundus, measuring intracellular calcium mobilization, and competitive fluorimetric binding assays. BK showed 2- and 15-fold decreased potency for Glu5.35(226) and Asp6.58(298) B2 mutant receptors, respectively. In B2-Glu5.35(226)Ala BK analogs showed milder reduction in evaluated parameters. On the other hand, in the B2-Asp6.58(298)Ala mutant, no N-terminal analog was able to elicit any response. However, the BK-A9 analog presented higher affinity parameters than BK in the latter mutant. These findings provide enough support for defining a novel interaction role of BK-R9 and Asp6.58(298) receptor residues.

Short peptide constructs mimic agonist sites of AT(1)R and BK receptors.

Extracellular peptide ligand binding sites, which bind the N-termini of angiotensin II (AngII) and bradykinin (BK) peptides, are located on the N-terminal and extracellular loop 3 regions of the AT(1)R and BKRB(1) or BKRB(2) G-protein-coupled receptors (GPCRs). Here we synthesized peptides P15 and P13 corresponding to these receptor fragments and showed that only constructs in which these peptides were linked by S-S bond, and cyclized by closing the gap between them, could bind agonists. The formation of construct-agonist complexes was revealed by electron paramagnetic resonance spectra and fluorescence measurements of spin labeled biologically active analogs of AngII and BK (Toac(1)-AngII and Toac(0)-BK), where Toac is the amino acid-type paramagnetic and fluorescence quencher 2, 2, 6, 6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid. The inactive derivatives Toac(3)-AngII and Toac(3)-BK were used as controls. The interactions characterized by a significant immobilization of Toac and quenching of fluorescence in complexes between agonists and cyclic constructs were specific for each system of peptide-receptor construct assayed since no crossed reactions or reaction with inactive peptides could be detected. Similarities among AT, BKR, and chemokine receptors were identified, thus resulting in a configuration for AT(1)R and BKRB cyclic constructs based on the structure of the CXCR(4), an α-chemokine GPCR-type receptor.

GPCRDB: information system for G protein-coupled receptors.

The GPCRDB is a Molecular Class-Specific Information System (MCSIS) that collects, combines, validates and disseminates large amounts of heterogeneous data on G protein-coupled receptors (GPCRs). The GPCRDB contains experimental data on sequences, ligand-binding constants, mutations and oligomers, as well as many different types of computationally derived data such as multiple sequence alignments and homology models. The GPCRDB provides access to the data via a number of different access methods. It offers visualization and analysis tools, and a number of query systems. The data is updated automatically on a monthly basis. The GPCRDB can be found online at http://www.gpcr.org/7tm/.

Role of the second disulfide bridge (Cys(18)-Cys(274)) in stabilizing the inactive AT1 receptor.

Previous research showed that disruption of the Cys(18)-Cys(274) bond in the angiotensin II (AngII) AT1 receptor mutant (C18S), expressed in CHO cells, causes an increase in the basal activity and attenuation of the maximum response to AngII. In addition, this mutant was mostly intracellularly distributed. Our aim was to investigate whether the intracellular presence of the mutant was due to a constitutive internalization or to a defective maturation of the receptor. The first hypothesis was assessed by pretreating the cells with losartan or [Sar¹Leu8]-AngII, specific AT1 receptor antagonists, a maneuver to revert the receptor internalization. The second hypothesis was tested using calnexin, an endoplasmic reticulum marker. We found that treatment with AT1 receptor antagonists causes an increase in the binding ability of the mutant to AngII. Furthermore, whereas the maximum effect is increased, it reduces the enhanced basal levels of IP3. The hypothesis for a lack of maturation of the mutant receptor was ruled out because calnexin was poorly colocalized with the intracellular C18S receptor. Our results suggest that the mutation of the AT1 receptor leads to a conformational structure similar to that of the active mode of the AT1 receptor, favoring its internalization in the absence of the agonist.

Carcinoma ductal em mama supranumérica / Ductal carcinoma in supernumerary breast

Relato de caso de uma paciente que teve diagnosticado um carcinoma infiltrante de mama extranumerária. Foram revisados os arquivos médicos, as peças de anatomia patológica e a literatura científica. Paciente feminina, 47 anos, foi encaminhada ao Serviço de Mastologia do Hospital Santa Casa de Misericórdia de Curitiba, em 2006, para investigação de nódulo sólido na transição dos quadrantes inferiores da mama esquerda demonstrado na mamografia. Ao exame físico, notava-se a presença de mama extranumerária com complexo aréolo-papilar em sulco inframamário esquerdo, sem nódulos palpáveis e expressão negativa. Axilas, fossas supraclaviculares e infraclaviculares livres. A core-biopsy da lesão revelou carcinoma ductal infiltrante moderadamente diferenciado com receptores hormonais positivos e CERB-B2 negativo. Foi indicada a mastectomia da mama exatranumerária esquerda com biópsia de linfonodo sentinela. O exame histopatológico definitivo revelou um carcinoma ductal infiltrante, moderadamente diferenciado, medindo 1,0 x 0,7 cm, invasão angiolinfática ausente, com dois linfonodos sentinela negativos. O tratamento escolhido foi realizado com radioterapia adjuvante exclusiva e hormonioterapia com tamoxifeno. Apesar da raridade, o câncer de mama ectópico merece atenção e cuidado por apresentar difícil diagnóstico e alta morbimortalidade.

Case report of a patient who was diagnosed with infiltrating breast carcinoma extranumerária. We reviewed the medical files, parts of anatomy and the scientific literature. Female patient, 47 years, was referred to the breast cancer unit at the Hospital Santa Casa de Misericordia de Curitiba, in 2006, for investigation of solid nodule in the transition of the lower quadrants of the left breast demonstrated by mammography. On physical examination, we noted the presence of breast extranumerária with papillary-areolar complex in the left inframammary fold, with no palpable nodules and negative expression. Axilla, supraclavicular and infraclavicular fossae free. The core-biopsy of the lesion revealed moderately differentiated infiltrating ductal carcinoma with hormone receptor positive and negative CERB-B2. Was indicated exatranumerária left breast mastectomy with sentinel lymph node biopsy. The final histopathology revealed a ductal carcinoma, moderately differentiated, measuring 1.0 x 0.7 cm, invasion angiolymphatic absent, with two sentinel nodes negative. The chosen treatment was performed with adjuvant radiotherapy and hormone therapy with tamoxifen alone. Despite its rarity, ectopic breast cancer deserves attention and care to present difficult diagnosis and high mortality.

Factors regulating tachyphylaxis triggered by N-terminal-modified angiotensin II analogs.

Binding of angiotensin II (DRVYIHPF, AngII) to its AT(1) receptor can trigger a process known as tachyphylaxis (loss of receptor response owing to repeated agonist stimulation). We propose a two-state binding model for tachyphylaxis where the N-terminal Asp(1) and Arg(2) residues of the peptide are supposed to initially bind to the N-terminal segment (Arg(23)) and to the EC-3 loop (Asp(281)) of an AT(1) molecule, respectively (state 1). Sequentially, a disruption of the salt bond between the AngII Asp(1) beta-carboxyl function and the receptor Arg(23) can occur with release of the peptide N-terminal segment, favoring the binding of the Arg(2) residue to the EC-3 loop (Asp(178,281), state 2). In the present study, we expanded this investigation by assaying pharmacological properties of different AngII analogs in guinea-pig ileum bearing modifications at positions 1 and 2. Most of these peptides were weak agonists but many of them had the ability to induce tachyphylaxis. These findings support the two-state model for tachyphylaxis, but alternative mechanisms were revealed where state 1 was no longer needed, depending on the chemical structure of AngII residue 1. Otherwise, any modification of the wild type AngII Arg(2) residue was deleterious for the tachyphylaxis mechanism.

Distinct binding mode of 125I-AngII to AT1 receptor without the Cys18-Cys274 disulfide bridge.

Previous studies on angiotensin II (AngII) AT(1) receptor function have revealed that the N-terminal residues of AngII may modulate receptor activation by binding at the receptor extracellular site. A remarkable feature of this site is an insertion of 8 amino acids in the middle of the EC-3 loop including the Cys(274) residue that supposedly makes a disulfide bond with N-terminal Cys(18). As demonstrated by assays with Del(267-275)AT(1), the role of the Cys(18)-Cys(274) disulfide bridge is to keep a conformation of the inserted residues that allows a normal binding of the AngII N-terminal residues. C18S AT(1) receptor mutant, supposedly having a dissociated disulfide bridge, but an intact residue insertion, is constitutively activated and can less efficiently bind AngII. Similar results were observed when the S-S disulfide bond was disrupted in (C18S,C274S) AT(1) receptor. The importance of the free N-terminal amino group of Asp(1) and of the Arg(2) guanidino group for the binding of AngII to C18S mutant with EC-3 loop insertion was investigated by means of assays using AngII peptide analogues bearing a single mutation of Asp(1) for Sar(1) or Arg(2) for Lys(2), as ligands. This study showed that like AngII, [Sar(1)]-AngII can bind the C18S mutant receptor with low affinity whereas [Lys(2)]-AngII binding is still more reduced. Interestingly, when (125)I-AngII instead of (3)H-AngII was used, no significant binding of this mutant was observed although wild type AT(1) receptor was shown to bind all AngII analogues.

Family A G-Protein coupled receptors: an overview of structure-function relationships

Family A G-protein coupled receptors (AGPCRs) form the largest group of correlate receptors whose structure, a bundle of seven-trans-membrane (7 TM) helices, may be activated thus becoming able to transduce a signal from the extracellular medium to the cytosol. This activation may be constitutional, for instance due to permanent structural modifications, or be physiologically triggered by agonist binding at an external and accessible specific site. Based on thestructures of agonists, AGPCRs may be divided according to pharmacological assays into many classes of receptors, each one comprising many types or sub-types of proteins, as differentiated by specific binding of inhibitors, all of them performing a multitude of functions. It is noteworthy that AGPCRs have been more recently cloned and their sequences of amino acids determined in a large scale, a condition that has allowed these receptors to be sorted by a new criterium. Sequence analyses have consistently matched functional assays for classification of AGPCRs except for a certain number of functionally unknown receptors which have been cataloged as orphan receptors. A colossal number of AGPCRs, more than 10,000 sequences belonging to more than 1,000 different types of receptors, may nowadays be multiply-aligned what has been enabling the determination of parameters of residue conservation and characterization of special motifs along the structure of these proteins. There are at the present time, high-resolution 3D structures for the following AGPCRs: inactive rhodopsin, retinal-free opsin, Beta adrenoceptor and adenosine receptors. Among them, hodopsin structures are reliable enough to be used as prototypes for analyses of residue conservation and mechanisms of activation of receptors, specially at the level of the more conserved structure in the cytosolic half of their 7TM bundle.

Accuracy of laparoscopy for assessing patients with endometriosis / Eficácia da videolaparoscopia na avaliação de mulheres com endometriose pélvica

CONTEXT AND OBJECTIVE: Diagnoses of endometriosis are based on observation of endometriotic lesions by means of laparoscopy, along with the pathological findings. The aim of this study was to evaluate the sensitivity and specificity of the macroscopic findings in relation to the histopathological findings. More specifically, we aimed to test the efficacy of laparoscopy alone for diagnosing endometriosis and to evaluate the laterality of endometriosis among the study population. DESIGN AND SETTING: Cross-sectional study on women undergoing laparoscopy due to pelvic pain or infertility, in the Gynecology Department of Hospital Santa Cruz in Curitiba, Paraná, Brazil, and Pontifícia Universidade Católica do Paraná. METHODS: A total of 976 patients underwent laparoscopy and biopsy due to pelvic pain and/or infertility. We analyzed the laparoscopic and histopathological findings from patients with pelvic endometriosis (n = 468) and patients without endometriosis (n = 508). RESULTS: In 468 (47.95 percent) of the cases, the clinical and laparoscopic findings were consistent with endometriosis, and this was confirmed histopathologically in 337 (34.5 percent). Among the remaining 508 patients, although the laparoscopy was performed for other reasons relating to acute pelvic pain, eight were diagnosed with endometriosis from histopathological examination of the pelvic specimens obtained. Therefore, endometriosis was confirmed in 345 patients (35.3 percent). In comparison with the histopathology, laparoscopy alone presented 97.68 percent sensitivity, 79.23 percent specificity, 72 percent positive predictive value and 98.42 percent negative predictive value. CONCLUSION: Laparoscopy should be used in conjunction with histopathology for diagnosing endometriosis.

CONTEXTO E OBJETIVO: O diagnóstico da endometriose é determinado pela visualização dos implantes à laparoscopia e pela comprovação histológica. O objetivo deste trabalho foi avaliar a sensibilidade e a especificidade dos achados macroscópicos cirúrgicos e histopatológicos. Avaliou-se a eficácia da laparoscopia isoladamente no diagnóstico da endometriose e a lateralidade da doença. TIPO DE ESTUDO E LOCAL: Estudo transversal realizado no Serviço de Ginecologia do Hospital Santa Cruz em Curitiba, Paraná e na Pontifícia Universidade Católica do Paraná. MÉTODOS: Foram avaliadas 976 pacientes submetidas à videolaparoscopia por dor pélvica ou infertilidade e a biópsia. Foram analisados os achados laparoscópicos e histológicos de 468 pacientes com endometriose pélvica e de 508 pacientes sem endometriose. RESULTADOS: Foram selecionadas 468 (47,95 por cento) pacientes para inclusão no presente estudo por apresentarem quadro clínico e videolaparoscópico de suspeita de endometriose. As 508 (52,04 por cento) pacientes restantes tiveram indicação da cirurgia por outras causas relacionadas à dor pélvica e oito tiveram o diagnóstico de endometriose pelo anatomopatológico. A endometriose foi confirmada em 345 pacientes (35,3 por cento). Ao compararmos a análise histológica com os achados a videolaparoscopia, observou-se sensibilidade de 97,68 por cento, especificidade de 79,23 por cento, valor preditivo positivo de 72 por cento, valor preditivo negativo de 98,42 por cento. CONCLUSÃO: Laparoscopia deve ser usada em conjunto com histopatologia para o diagnóstico de endometriose.

Essential role of TM V and VI for binding the C-terminal sequences of Des-Arg-kinins.

In the kallikrein-kinin and renin-angiotensin systems the main receptors, B1 and B2 (kinin receptors) and AT1 and AT2 (angiotensin receptors) respectively, are seven-transmembrane domain G-protein-coupled receptors. Considering that the B1 agonists Des-Arg9-BK (Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe), Lys-desArg9-BK or Des-Arg10-KD (Lys-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe) and the AT1 agonist (Asp-Arg-Val-Tyr-Ile-His-Pro-Phe) have the same two residues at the C-terminal region (i.e. Pro-Phe), we hypothesized that TM V and TM VI of the B1 receptor could play an essential role in agonist binding and activity, being these regions receptor sites for binding the C-terminal sequences of Des-Arg-kinins similarly to that observed to AT1 receptor. To investigate this hypothesis, we replaced Arg212 for Ala at the top of the TM V and the sequence 274-282 (CPYHFFAFL) in TM VI of the rat kinin B1 receptor by the B2 receptor homologous sequence, 289-297 (FPFQISTFL) and subsequently analyzed the consequences of these mutations by competition binding and functional assays. Despite correct expression, observed at the mRNA and protein level by RT-PCR and confocal microscopy, respectively, no agonist binding and function was verified for the mutated receptors. Therefore, our results suggest an important role for Arg212 in the TM V and a region of TM VI of rat B1 receptor in the interaction with the C-terminal residues of Des-Arg-kinins, similar to that observed with AngII.

Accuracy of laparoscopy for assessing patients with endometriosis.

CONTEXT AND OBJECTIVE: Diagnoses of endometriosis are based on observation of endometriotic lesions by means of laparoscopy, along with the pathological findings. The aim of this study was to evaluate the sensitivity and specificity of the macroscopic findings in relation to the histopathological findings. More specifically, we aimed to test the efficacy of laparoscopy alone for diagnosing endometriosis and to evaluate the laterality of endometriosis among the study population. DESIGN AND SETTING: Cross-sectional study on women undergoing laparoscopy due to pelvic pain or infertility, in the Gynecology Department of Hospital Santa Cruz in Curitiba, Paraná, Brazil, and Pontifícia Universidade Católica do Paraná. METHODS: A total of 976 patients underwent laparoscopy and biopsy due to pelvic pain and/or infertility. We analyzed the laparoscopic and histopathological findings from patients with pelvic endometriosis (n = 468) and patients without endometriosis (n = 508). RESULTS: In 468 (47.95%) of the cases, the clinical and laparoscopic findings were consistent with endometriosis, and this was confirmed histopathologically in 337 (34.5%). Among the remaining 508 patients, although the laparoscopy was performed for other reasons relating to acute pelvic pain, eight were diagnosed with endometriosis from histopathological examination of the pelvic specimens obtained. Therefore, endometriosis was confirmed in 345 patients (35.3%). In comparison with the histopathology, laparoscopy alone presented 97.68% sensitivity, 79.23% specificity, 72% positive predictive value and 98.42% negative predictive value. CONCLUSION: Laparoscopy should be used in conjunction with histopathology for diagnosing endometriosis.

The angiotensin II AT1 receptor structure-activity correlations in the light of rhodopsin structure.

The most prevalent physiological effects of ANG II, the main product of the renin-angiotensin system, are mediated by the AT1 receptor, a rhodopsin-like AGPCR. Numerous studies of the cardiovascular effects of synthetic peptide analogs allowed a detailed mapping of ANG II's structural requirements for receptor binding and activation, which were complemented by site-directed mutagenesis studies on the AT1 receptor to investigate the role of its structure in ligand binding, signal transduction, phosphorylation, binding to arrestins, internalization, desensitization, tachyphylaxis, and other properties. The knowledge of the high-resolution structure of rhodopsin allowed homology modeling of the AT1 receptor. The models thus built and mutagenesis data indicate that physiological (agonist binding) or constitutive (mutated receptor) activation may involve different degrees of expansion of the receptor's central cavity. Residues in ANG II structure seem to control these conformational changes and to dictate the type of cytosolic event elicited during the activation. 1) Agonist aromatic residues (Phe8 and Tyr4) favor the coupling to G protein, and 2) absence of these residues can favor a mechanism leading directly to receptor internalization via phosphorylation by specific kinases of the receptor's COOH-terminal Ser and Thr residues, arrestin binding, and clathrin-dependent coated-pit vesicles. On the other hand, the NH2-terminal residues of the agonists ANG II and [Sar1]-ANG II were found to bind by two distinct modes to the AT1 receptor extracellular site flanked by the COOH-terminal segments of the EC-3 loop and the NH2-terminal domain. Since the [Sar1]-ligand is the most potent molecule to trigger tachyphylaxis in AT1 receptors, it was suggested that its corresponding binding mode might be associated with this special condition of receptors.

Functional rescue of a defective angiotensin II AT1 receptor mutant by the Mas protooncogene.

Earlier studies with Mas protooncogene, a member of the G-protein-coupled receptor family, have proposed this gene to code for a functional AngII receptor, however further results did not confirm this assumption. In this work we investigated the hypothesis that a heterodimeration AT(1)/Mas could result in a functional interaction between both receptors. For this purpose, CHO or COS-7 cells were transfected with the wild-type AT(1) receptor, a non-functional AT(1) receptor double mutant (C18F-K20A) and Mas or with WT/Mas and C18F-K20A/Mas. Cells single-expressing Mas or C18F/K20A did not show any binding for AngII. The co-expression of the wild-type AT(1) receptor and Mas showed a binding profile similar to that observed for the wild-type AT(1) expressed alone. Surprisingly, the co-expression of the double mutant C18F/K20A and Mas evoked a total recovery of the binding affinity for AngII to a level similar to that obtained for the wild-type AT(1). Functional measurements using inositol phosphate and extracellular acidification rate assays also showed a clear recovery of activity for AngII on cells co-expressing the mutant C18F/K20A and Mas. In addition, immunofluorescence analysis localized the AT(1) receptor mainly at the plasma membrane and the mutant C18F-K20A exclusively inside the cells. However, the co-expression of C18F-K20A mutant with the Mas changed the distribution pattern of the mutant, with intense signals at the plasma membrane, comparable to those observed in cells expressing the wild-type AT(1) receptor. These results support the hypothesis that Mas is able to rescue binding and functionality of the defective C18F-K20A mutant by dimerization.

Participation of transmembrane proline 82 in angiotensin II AT1 receptor signal transduction.

Most of the classical physiological effects of the octapeptide angiotensin II (AngII) are produced by activating the AT1 receptor which belongs to the G-protein coupled receptor family (GPCR). Peptidic GPCRs may be functionally divided in three regions: (i) extracellular domains involved in ligand binding; (ii) intracellular domains implicated in agonist-induced coupling to G protein and (iii) seven transmembrane domains (TM) involved in signal transduction. The TM regions of such receptors have peculiar characteristics such as the presence of proline residues. In this project we aimed to investigate the participation of two highly conserved proline residues (Pro82 and Pro162), located in TM II and TM IV, respectively, in AT1 receptor signal transduction. Both mutations did not cause major alterations in AngII affinity. Functional assays indicated that the P162A mutant did not influence the signal transduction. On the other hand, a potent deleterious effect of P82A mutation on signal transduction was observed. We believe that the Pro82 residue is crucial to signal transduction, although it is not possible to say yet if this is due to a direct participation or if due to a structural rearrangement of TM II. In this last hypothesis, the removal of proline residue might be correlated to a removal of a kink, which in turn can be involved in the correct positioning of residues involved in signal transduction.

Molecular determinants for G protein betagamma modulation of ionotropic glycine receptors.

The ligand-gated ion channel superfamily plays a critical role in neuronal excitability. The functions of glycine receptor (GlyR) and nicotinic acetylcholine receptor are modulated by G protein betagamma subunits. The molecular determinants for this functional modulation, however, are still unknown. Studying mutant receptors, we identified two basic amino acid motifs within the large intracellular loop of the GlyR alpha(1) subunit that are critical for binding and functional modulation by Gbetagamma. Mutations within these sequences demonstrated that all of the residues detected are important for Gbetagamma modulation, although both motifs are necessary for full binding. Molecular modeling predicts that these sites are alpha-helixes near transmembrane domains 3 and 4, near to the lipid bilayer and highly electropositive. Our results demonstrate for the first time the sites for G protein betagamma subunit modulation on GlyRs and provide a new framework regarding the ligand-gated ion channel superfamily regulation by intracellular signaling.

Molecular modeling of the human eukaryotic translation initiation factor 5A (eIF5A) based on spectroscopic and computational analyses.

The eukaryotic translation initiation factor 5A (eIF5A) is a protein ubiquitously present in archaea and eukarya, which undergoes a unique two-step post-translational modification called hypusination. Several studies have shown that hypusination is essential for a variety of functional roles for eIF5A, including cell proliferation and synthesis of proteins involved in cell cycle control. Up to now neither a totally selective inhibitor of hypusination nor an inhibitor capable of directly binding to eIF5A has been reported in the literature. The discovery of such an inhibitor might be achieved by computer-aided drug design based on the 3D structure of the human eIF5A. In this study, we present a molecular model for the human eIF5A protein based on the crystal structure of the eIF5A from Leishmania brasiliensis, and compare the modeled conformation of the loop bearing the hypusination site with circular dichroism data obtained with a synthetic peptide of this loop. Furthermore, analysis of amino acid variability between different human eIF5A isoforms revealed peculiar structural characteristics that are of functional relevance.

Role of the Cys18-Cys274 disulfide bond and of the third extracellular loop in the constitutive activation and internalization of angiotensin II type 1 receptor.

An insertion of residues in the third extracellular loop and a disulfide bond linking this loop to the N-terminal domain were identified in a structural model of a G-protein coupled receptor specific to angiotensin II (AT1 receptor), built in homology to the seven-transmembrane-helix bundle of rhodopsin. Both the insertion and the disulfide bond were located close to an extracellular locus, flanked by the second extracellular loop (EC-2), the third extracellular loop (EC-3) and the N-terminal domain of the receptor; they contained residues identified by mutagenesis studies to bind the angiotensin II N-terminal segment (residues D1 and R2). It was postulated that the insertion and the disulfide bond, also found in other receptors such as those for bradykinin, endothelin, purine and other ligands, might play a role in regulating the function of the AT1 receptor. This possibility was investigated by assaying AT1 forms devoid of the insertion and with mutations to Ser on both positions of Cys residues forming the disulfide bond. Binding and activation experiments showed that abolition of this bond led to constitutive activation, decay of agonist binding and receptor activation levels. Furthermore, the receptors thus mutated were translocated to cytosolic environments including those in the nucleus. The receptor form with full deletion of the EC-3 loop residue insertion, displayed a wild type receptor behavior.

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.

Angiotensin II AT1 receptor mutants expressed in CHO cells caused morphological change and inhibition of cell growth.

To assess the importance of the leucine residues in positions 262 and 265 of the angiotensin AT(1) receptor for signaling pathways and receptor expression and regulation, we compared the properties of CHO cells transfected with the wild type or the L262D or L265D receptor point mutants. It was found that the two mutants significantly increased the basal intracellular cyclic AMP (cAMP) formation in an agonist-independent mode. The morphology transformation of CHO cells was correlated with the increased cAMP formation, since forskolin, a direct activator of adenylate cyclase mimicked this effect on WT-expressing CHO cells. DNA synthesis was found to be inhibited in these cell lines, indicating that cAMP may also have determined the inhibitory effect on cell growth, in addition to the cell transformation from a tumorigenic to a non-tumorigenic phenotype. However a role for an increased Ca2+ influx induced by the mutants in non-stimulated cells cannot be ruled out since this ion also was shown to cause transformed cells to regain the morphology and growth regulation.

A family-based approach reveals the function of residues in the nuclear receptor ligand-binding domain.

Literature studies, 3D structure data, and a series of sequence analysis techniques were combined to reveal important residues in the structure and function of the ligand-binding domain of nuclear hormone receptors. A structure-based multiple sequence alignment allowed for the seamless combination of data from many different studies on different receptors into one single functional model. It was recently shown that a combined analysis of sequence entropy and variability can divide residues in five classes; (1) the main function or active site, (2) support for the main function, (3) signal transduction, (4) modulator or ligand binding and (5) the rest. Mutation data extracted from the literature and intermolecular contacts observed in nuclear receptor structures were analyzed in view of this classification and showed that the main function or active site residues of the nuclear receptor ligand-binding domain are involved in cofactor recruitment. Furthermore, the sequence entropy-variability analysis identified the presence of signal transduction residues that are located between the ligand, cofactor and dimer sites, suggesting communication between these regulatory binding sites. Experimental and computational results agreed well for most residues for which mutation data and intermolecular contact data were available. This allows us to predict the role of the residues for which no functional data is available yet. This study illustrates the power of family-based approaches towards the analysis of protein function, and it points out the problems and possibilities presented by the massive amounts of data that are becoming available in the "omics era". The results shed light on the nuclear receptor family that is involved in processes ranging from cancer to infertility, and that is one of the more important targets in the pharmaceutical industry.