Laerte Oliveira

A common motif in G-protein-coupled seven transmembrane helix receptors

SummaryG-protein-coupled receptors all share the seven transmembrane helix motif similar to bacteriorhodopsin. This similarity was exploited to build models for these receptors. From an analysis of a multi-sequence alignment of 225 G-protein-coupled receptors belonging to the rhodopsin-like superfamily, conclusions could be drawn about functional residues. Seven residues in the transmembrane regions are conserved throughout all aligned receptors. These residues cluster at the cytosolic side of the transmembrane helices and are for all rhodopsin-like G-protein-coupled receptors implied in signal transduction. An analysis of correlated mutations reveals a number of residues, both in the helices and in the cytosolic loops, that might be important in the signal transduction pathway in subfamilies of this receptor family.

Receptors coupling to G proteins: is there a signal behind the sequence?

Upon the binding of their ligands, G protein‐coupled receptors couple to the heterotrimeric G proteins to transduce a signal. One receptor family may couple to a single G protein subtype and another family to several ones. Is there a signal in the receptor sequence that can give an indication of the G protein subtype selectivity? We used a sequence analysis method on biogenic amine and adenosine receptors and concluded that a weak signal can be detected in receptor families where specialization for coupling to a given G protein occurred during a recent divergent evolutionary process. Proteins 2000;41:448–459.

Identification of Functionally Conserved Residues With the Use of Entropy-Variability Plots

We introduce sequence entropy–variability plots as a method of analyzing families of protein sequences, and demonstrate this for three well‐known sequence families: globins, ras‐like proteins, and serine‐proteases. The location of an aligned residue position in the entropy–variability plot correlates with structural characteristics, and with known facts about the roles of individual amino acids in the function of these proteins. The large numbers of known sequences in these families allowed us to introduce new filtering methods for variability patterns. The results are discussed in terms of a simple evolutionary model for functional proteins. Proteins 2003;52:544–552.

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.

Sequence analysis reveals how G protein-coupled receptors transduce the signal to the G protein.

Sequence entropy—variability plots based on alignments of very large numbers of sequences—can indicate the location in proteins of the main active site and modulator sites. In the previous article in this issue, we applied this observation to a series of well‐studied proteins and concluded that it was possible to detect most of the residues with a known functional role. Here, we apply the method to rhodopsin‐like G protein–coupled receptors. Our conclusion is that G protein binding is the main evolutionary constraint on these receptors, and that other ligands, such as agonists, act as modulators. The activation of the receptors can be described as a simple, two‐step process, and the residues involved in signal transduction can be identified. Proteins 2003;52:553–560.

Mutagenesis of the AT1 receptor reveals different binding modes of angiotensin II and [Sar1]-angiotensin II

Homology modeling of the structure of the AT1 receptor, based on the high resolution rhodopsin crystal structure, indicated that it is unlikely that the binding of AngII to AT1 involves simultaneously all the receptors residues reported in the literature to participate in this process. Site-directed mutagenesis using Ala substitution of charged residues Lys20, Arg23, Glu91 and Arg93 was performed to evaluate the participation of their side-chains in ligand binding and in triggering the cells response. A comparative analysis by competition binding and functional assays using angiotensin II and the analog [Sar1]-angiotensin II suggests an important role for Arg23 of AT1 receptor in binding of the natural agonist. It is discussed whether some receptors residues participate directly in the binding with AngII or whether they are part of a regulatory site.

Mutational analysis of the interaction of the N‐ and C‐terminal ends of angiotensin II with the rat AT1A receptor

The role of different residues of the rat AT1A receptor in the interaction with the N‐ and C‐terminal ends of angiotensin II (AngII) was studied by determining ligand binding and production of inositol phosphates (IP) in COS‐7 cells transiently expressing the following AT1A mutants: T88H, Y92H, G196I, G196W and D278E. G196W and G196I retained significant binding and IP‐production properties, indicating that bulky substituents in position 196 did not affect the interaction of AngIIs C‐terminal carboxyl with Lys199 located three residues below. Although the T88A mutation did not affect binding, the T88H mutant had greatly decreased affinity for AngII, suggesting that substitution of Thr88 by His might hinder binding through an indirect effect. The Y92H mutation caused loss of affinity for AngII that was much less pronounced than that reported for Y92A, indicating that His in that position can fulfil part of the requirements for binding. Replacing Asp278 by Glu caused a much smaller reduction in affinity than replacing it by Ala, indicating the importance of Asps β‐carboxyl group for AngII binding. Mutations in residues Thr88, Tyr92 and Asp278 greatly reduced affinity for AngII but not for Sar1 Leu8‐AngII, suggesting unfavourable interactions between these residues and AngIIs aspartic acid side‐chain or N‐terminal amino group, which might account for the proposed role of the N‐terminal amino group of AngII in the agonist‐induced desensitization (tachyphylaxis) of smooth muscles.

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.

Aliphatic amino acids in helix VI of the AT1 receptor play a relevant role in agonist binding and activity

Angiotensin II (AII) AT(1) receptor mutants with replacements of aliphatic amino acids in the distal region of helix VI and the adjoining region of the third extracellular loop (EC-3) were expressed in Chinese hamster ovary (CHO) cells to determine their role in ligand binding and activation. The triple mutant [L262D, L265D, L268D]AT(1) (L3D) showed a marked reduction in affinity for AII and for non-peptide (losartan) and peptide ([Sar(1)Leu(8) ]AII) antagonists; in functional assays using inositol phosphate (IP) accumulation, the relative potency and the maximum effect of AII were reduced in L3D. Replacement of Leu(268) (in EC-3) and Leu(262) (in the transmembrane domain) by aspartyl residues did not cause significant changes in the receptors affinity for the ligands and in IP production. In contrast, the point mutation L265D, at helix VI, markedly decreased affinity and ability to stimulate phosphatidylinositol turnover. Molecular modeling of the AT(1) receptor based on a recent crystal structure of rhodopsin, suggests that the side chain of Leu(265) but not that of Leu(262) is facing a cleft between helices V and VI and interacts with the lipid bilayer, thus helping to stabilize the receptor structure near the Lys(199) residue of helix V in the agonist binding site which is necessary for full activity.

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.