Antonio C. M. Paiva

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.

Altered neutrophil homeostasis in kinin B1 receptor-deficient mice.

Abstract The kallikrein-kinin system is activated during inflammation and plays a major role in the inflammatory process. One of the main mechanisms of kinin action includes the modulation of neutrophil function employing both receptors for kinins, B1 and B2. In this report we show by the use of B1 receptor-deficient mice that neutrophil migration in inflamed tissues is dependent on kinin B1 receptors. However, there is no change in circulating leukocyte number and composition after genetic ablation of this receptor. Furthermore, apoptosis of neutrophils necessary for the resolution of persistent inflammatory processes is impaired in mice lacking the B1 receptor. We also show that this receptor is expressed on neutrophils, thus it may be directly involved in the induction of apoptosis in these cells after prolonged activation at inflamed sites. In conclusion, our data show that the kinin B1 receptor modulates migration and the life span of neutrophils at sites of inflammation and may be therefore an important drug target in the therapy of inflammatory diseases.

Further evidence for the existence of two receptor sites for bradykinin responsible for the diphasic effect in the rat isolated duodenum

1 Low doses of bradykinin (below 10 nM), as well as of K+ (below 10 mM) induced relaxation, whereas higher doses caused contraction of the rat duodenum. 2 The relaxant responses induced by bradykinin and K+ were not affected by ouabain (1 μM), but pre‐incubation with 5.9 mM K+ abolished the responses to that ion but not those to bradykinin. 3 The contractile and relaxant components of the response to bradykinin (but not those to K+) increased with the time elapsed after mounting of the preparation, and this was due to stretching by the load of the recording system. 4 Specific and reversible desensitization (tachyphylaxis) was observed with the contractile response (but not the relaxation) induced by bradykinin. 5 Des‐Arg9‐bradykinin, an analogue specific for B1‐receptors, was much less active than bradykinin, and elicited only a contractile response. 6 Among four bradykinin potentiating peptides that were tested, potentiator C enhanced the relaxation only, whereas BPP5a and captopril potentiated only the contraction and BPP9a potentiated both types of response to bradykinin. 7 Our results support the hypothesis that the relaxant and contractile components of the rat duodenums response to bradykinin are due to actions at different receptor sites, which can be distinguished by their properties (desensitization) and their different apparent affinities for agonists and for potentiating peptides.

Bradykinin metabolism pathway in the rat pulmonary circulation.

Objective: The contribution made by different enzymes to the degradation of bradykinin in physiological conditions was estimated by examining bradykinin metabolism in rat serum, in the in situ perfused lung and in vivo. Methods: Dose—response curves for the hypotensive effect of intra‐arterially and intravenously injected bradykinin were obtained in unanaesthetized rats. High‐performance liquid chromatography was used to analyse the products of bradykinin breakdown after incubation with rat serum and perfusion through in situ lung preparations. Results: In rat serum, kininase I degraded 34% and kininase II 11% of bradykinin, no evidence for other activities being detected. In the awake rat, D,L‐2‐mercaptomethyl‐3‐guanidino‐ethylthiopropionic acid, an inhibitor of kininase I, did not reduce the percentage of bradykinin inactivation in the pulmonary circulation. In the in situ perfused lung 65% of bradykinin was metabolized and the main products were BK1‐7, BK1‐5 and BK4‐9. Enalaprilat (an inhibitor of kininase II) blocked the formation of BK1‐7 and BK1‐5 and increased the recovery of BK4‐9. &bgr;‐Mercapto‐ethanol, which inhibits aminopeptidase P, and diprotin A, a specific inhibitor of dipeptidylaminopeptidase IV, both reduced the formation of BK4‐9. Diprotin A also allowed the recovery of BK2‐9. Bradykinin degradation and BK4‐9 recovery were not affected by endopeptidase inhibitors. Conclusions: Our results show that the main degradation pathway of bradykinin in the lung is through the action of kininase II at the carboxyl terminus, and sequential cleavage by aminopeptidase P followed by dipeptidylaminopeptidase IV at the amino terminus. The amino‐terminal degradation of bradykinin represents about 38% of the total lung kininase activity.

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.

Structure of Two Fragments of the Third Cytoplasmic Loop of the Rat Angiotensin II AT1A Receptor IMPLICATIONS WITH RESPECT TO RECEPTOR ACTIVATION AND G-PROTEIN SELECTION AND COUPLING

The structural bases that render the third intracellular loop (i3) of the rat angiotensin II AT1Areceptor one of the cytoplasmic domains responsible for G-protein coupling are still unknown. The three-dimensional structures of two overlapping peptides mapping the entire i3 loop and shown to differently interact with purified G-proteins have been obtained by simulated annealing calculations, using NMR-derived constraints collected in 70% water/30% trifluoroethanol solution. While the NH2-terminal half, Ni3, residues 213–231, adopts a stable amphipathic α-helix, extending over almost the entire peptide, a more flexible conformation is found for the COOH-terminal half, Ci3, residues 227–242. For this peptide, a cis-transisomerization around the Lys6—Pro7 peptide bond generates two exchanging isomers adopting similar conformations, with an α-helix spanning from Asn9 to Ile15and a poorly defined NH2 terminus. A quite distinct structural organization is found for the sequence EIQKN, common to Ni3 and Ci3. The data do suggest that the extension and orientation of the amphipathic α-helix, present in the proximal part of i3, may be modulated by the distal part of the loop itself through the Pro233 residue. A molecular model where this possibility is considered as a mechanism for G-protein selection and coupling is presented.

Role of the two n-terminal residues of angiotensin II in the production of tachyphylaxis

The structural requirements for the production of angiotensin tachyphylaxis in the guinea-pig ileum were studied by analyzing the tachyphylactic properties of the following synthetic analogues of angiotensin II (AII): [1-sarcosine]AII, [1-betaine]AII; [1-guanidinoacetic]AII; betainyl-AII; [2-lysine]AII; [2-ornithine]AII. In the non-atropinized ileum, no tachyphylaxis was observed with any of the following analogues: [2-lysine]AII, [2-ornithine]AII, [2-ornithine]AII, [1-betaine]AII and betainyl-AII. [1-Guanidinoacetic]AII induced tachyphylaxis, but to a smaller degree than AII, while [1-sarcosine]AII was significantly more tachyphylactic than AII. Similar results were obtained in the atropinized ileum, except that moderate tachyphylaxis was also observed with betainyl-AII and [1-betaine]AII. The analogues with lysine or ornithine residues in position 2 did not induce tachyphylaxis under any of the conditions studied. It is concluded that, besides the protonated N-terminal amino group, the guanidino group of the Arg2 side-chain is essential for the manifestation of angiotensin tachyphylaxis in the guinea-pig ileum.

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.

The effect of pH on tachyphylaxis to angiotensin peptides in the isolated guinea pig ileum and rat uterus

Abstract Angiotensin tachyphylaxis in the isolated guinea pig ileum and rat uterus was studied with the aid of the following synthetic peptides: [Ile5]agiotensin II (A II), [Asn1] A II, [Gly1] A II, [Arg1] A II, des-Asp1-A II, [Gly1,Gly2] A II, [Suc1] A II and acetyl-A II. The pK values for the amino and imidazole groups of these peptides were determined by electrometric titration. The effect of pH on tachyphylaxis to the angiotensin analogs and homologs was studied in the two smooth muscle preparations. The intensity of tachyphylaxis produced by the peptides correlated well with the degree of protonation of the amino group, but not with that of the imidazole group or with the net charge of the peptide. [Gly1,Gly2] A II and the two peptides in which the amino group was absent ([Suc1] A II) or blocked (acetyl-A II) did not produce any tachyphylaxis in the guinea pig ileum or the rat uterus. It is concluded that the protonated amino group plays an important role in the phenomenon of tachyphylaxis, in both organs, and that the guanido group is also important.