Xavier Iturrioz

Functional dissociation of apelin receptor signaling and endocytosis: implications for the effects of apelin on arterial blood pressure

Apelin is a novel neuropeptide involved in the regulation of body fluid homeostasis and cardiovascular functions. It acts through a G protein‐coupled receptor, the APJ receptor. We studied the structure–activity relationships of apelin at the rat apelin receptor, tagged at its C‐terminal end with enhanced green fluorescent protein and stably expressed in CHO cells. We evaluated the potency of N‐ and C‐terminal deleted fragments of K17F to bind with high affinity to the apelin receptor, and to inhibit cAMP production and to induce apelin receptor internalization. We first characterized the internalization and trafficking of the rat apelin receptor. This receptor was internalized via a clathrin‐dependent mechanism and our results suggest that receptor trafficking may follow a recycling pathway. We then tried to identify the amino acids of K17F required for apelin activity. The first five N‐terminal and the last two C‐terminal amino acids of K17F were not essential for apelin binding or the inhibition of cAMP production. However, the full‐length sequence of K17F was the most potent inducer of apelin receptor internalization because successive N‐terminal amino‐acid deletions progressively reduced internalization and the removal of a single amino acid at the C‐terminus abolished this process. Finally, the most novel observation of this work is that hypotensive actions of apelin peptides correlate best with the ability of those ligands to internalize. Thus, apelin receptor signaling and endocytosis are functionally dissociated, possibly reflecting the existence of several conformational states of this receptor, stabilized by the binding of different apelin fragments to the apelin receptor.

Identification and pharmacological properties of E339-3D6, the first nonpeptidic apelin receptor agonist

Apelin plays a prominent role in body fluid and cardiovascular homeostasis. To explore further upstream the role played by this peptide, nonpeptidic agonists and antagonists of the apelin receptor are required. To identify such compounds that do not exist to date, we used an original fluorescence resonance energy transfer‐based assay to screen a G‐protein‐coupled receptor‐focused library of fluorescent compounds on the human EGFP‐tagged apelin receptor. This led to isolated E339–3D6 that displayed a 90 nM affinity and behaved as a partial agonist with regard to cAMP production and as a full agonist with regard to apelin receptor internalization. Finally, E339–3D6 induced vasorelaxation of rat aorta precontracted with noradrenaline and potently inhibited systemic vasopressin release in water‐deprived mice when intracerebroventricularly injected. This compound represents the first nonpeptidic agonist of the apelin receptor, the optimization of which will allow development of a new generation of vasodilator and aquaretic agents.—Iturrioz, X., Alvear‐Perez, R., De Mota, N., Franchet, C., Guillier, F., Leroux, V., Dabire, H., Le Jouan, M., Chabane, H., Gerbier, R., Bonnet, D., Berdeaux, A., Maigret, B., Galzi J.‐L., Hibert, M., Llorens‐Cortes, C. Identification and pharmacological properties of E339–3D6, FASEB J. 24, 1506–1517 (2010). www.fasebj.org

Reciprocal Regulation of Plasma Apelin and Vasopressin by Osmotic Stimuli

Apelin is a neuropeptide that co-localizes with vasopressin (AVP) in magnocellular neurons and is involved in body fluid homeostasis. Osmotic stimuli have opposite effects on the regulation of apelin and AVP secretion in animal models, but whether this is true in humans is unknown. This study investigated the relationship among osmolality, apelin, and AVP in 10 healthy men after infusion of hypertonic saline or loading with water to increase and decrease plasma osmolality, respectively. Increasing plasma osmolality was accompanied by a parallel, linear increase in plasma AVP concentration and by a decrease in plasma apelin concentration. In contrast, decreasing plasma osmolality by water loading reduced plasma AVP concentration and rapidly increased plasma apelin concentration. These findings suggest that regulation of apelin secretion contributes to the maintenance of body fluid homeostasis.

By interacting with the C-terminal Phe of apelin, Phe255 and Trp259 in helix VI of the apelin receptor are critical for internalization.

Apelin is the endogenous ligand of the orphan seven-transmembrane domain (TM) G protein-coupled receptor APJ. Apelin is involved in the regulation of body fluid homeostasis and cardiovascular functions. We previously showed the importance of the C-terminal Phe of apelin 17 (K17F) in the hypotensive activity of this peptide. Here, we show either by deleting the Phe residue (K16P) or by substituting it by an Ala (K17A), that it plays a crucial role in apelin receptor internalization but not in apelin binding or in Gαi-protein coupling. Then we built a homology three-dimensional model of the human apelin receptor using the cholecystokinin receptor-1 model as a template, and we subsequently docked K17F into the binding site. We visualized a hydrophobic cavity at the bottom of the binding pocket in which the C-terminal Phe of K17F was embedded by Trp152 in TMIV and Trp259 and Phe255 in TMVI. Using molecular modeling and site-directed mutagenesis studies, we further showed that Phe255 and Trp259 are key residues in triggering receptor internalization without playing a role in apelin binding or in Gαi-protein coupling. These findings bring new insights into apelin receptor activation and show that Phe255 and Trp259, by interacting with the C-terminal Phe of the pyroglutamyl form of apelin 13 (pE13F) or K17F, are crucial for apelin receptor internalization.

Contribution of Molecular Modeling and Site-directed Mutagenesis to the Identification of Two Structural Residues, Arg-220 and Asp-227, in Aminopeptidase A

Aminopeptidase A is a zinc metalloenzyme involved in the formation of brain angiotensin III, which exerts a tonic stimulatory action on the central control of blood pressure. Thus, central inhibitors of aminopeptidase A constitute putative central antihypertensive agents. Mutagenic studies have been performed to investigate organization of the aminopeptidase A active site, with a view to designing such inhibitors. The structure of one monozinc aminopeptidase (leukotriene A4 hydrolase) was recently resolved and used to construct a three-dimensional model of the aminopeptidase A ectodomain. This new model, highly consistent with the results of mutagenic studies, showed a critical structural interaction between two conserved residues, Arg-220 and Asp-227. Mutagenic replacement of either of these two residues disrupted maturation and subcellular localization and abolished the enzymatic activity of aminopeptidase A, confirming the critical structural role of these residues. In this study, we generated the first three-dimensional model of a strict aminopeptidase, aminopeptidase A. This model constitutes a new tool to probe further the active site of aminopeptidase A and to design new inhibitors of this enzyme.

Biased Signaling Favoring Gi over β-Arrestin Promoted by an Apelin Fragment Lacking the C-terminal Phenylalanine

Background: Apelin receptor represents a therapeutic target for cardiovascular diseases. Results: Apelin 17 activates ERK1/2 in a β-arrestin-dependent and G protein-dependent manner, whereas apelin 17 with deleted C-terminal phenylalanine only signals through the G protein. Conclusion: Biased signaling promoted by an apelin fragment lacking the C-terminal phenylalanine is favoring Gi over β-arrestin. Significance: Apelin receptor β-arrestin signaling may account for apelin hypotensive activity. Apelin plays a prominent role in body fluid and cardiovascular homeostasis. We previously showed that the C-terminal Phe of apelin 17 (K17F) is crucial for triggering apelin receptor internalization and decreasing blood pressure (BP) but is not required for apelin binding or Gi protein coupling. Based on these findings, we hypothesized that the important role of the C-terminal Phe in BP decrease may be as a Gi-independent but β-arrestin-dependent signaling pathway that could involve MAPKs. For this purpose, we have used apelin fragments K17F and K16P (K17F with the C-terminal Phe deleted), which exhibit opposite profiles on apelin receptor internalization and BP. Using BRET-based biosensors, we showed that whereas K17F activates Gi and promotes β-arrestin recruitment to the receptor, K16P had a much reduced ability to promote β-arrestin recruitment while maintaining its Gi activating property, revealing the biased agonist character of K16P. We further show that both β-arrestin recruitment and apelin receptor internalization contribute to the K17F-stimulated ERK1/2 activity, whereas the K16P-promoted ERK1/2 activity is entirely Gi-dependent. In addition to providing new insights on the structural basis underlying the functional selectivity of apelin peptides, our study indicates that the β-arrestin-dependent ERK1/2 activation and not the Gi-dependent signaling may participate in K17F-induced BP decrease.

A new strategy for treating hypertension by blocking the activity of the brain renin-angiotensin system with aminopeptidase A inhibitors.

Hypertension affects one-third of the adult population and is a growing problem due to the increasing incidence of obesity and diabetes. Brain RAS (renin-angiotensin system) hyperactivity has been implicated in the development and maintenance of hypertension in several types of experimental and genetic hypertension animal models. We have identified in the brain RAS that APA (aminopeptidase A) and APN (aminopeptidase N), two membrane-bound zinc metalloproteases, are involved in the metabolism of AngII (angiotensin II) and AngIII (angiotensin III) respectively. The present review summarizes the main findings suggesting that AngIII plays a predominant role in the brain RAS in the control of BP (blood pressure). We first explored the organization of the APA active site by site-directed mutagenesis and molecular modelling. The development and the use in vivo of specific and selective APA and APN inhibitors EC33 and PC18 respectively, has allowed the demonstration that brain AngIII generated by APA is one of the main effector peptides of the brain RAS, exerting a tonic stimulatory control over BP in conscious hypertensive rats. This identified brain APA as a potential therapeutic target for the treatment of hypertension, which has led to the development of potent orally active APA inhibitors, such as RB150. RB150 administered orally in hypertensive DOCA (deoxycorticosteroneacetate)-salt rats or SHRs (spontaneously hypertensive rats) crosses the intestinal, hepatic and blood-brain barriers, enters the brain, generates two active molecules of EC33 which inhibit brain APA activity, block the formation of brain AngIII and normalize BP for several hours. The decrease in BP involves two different mechanisms: a decrease in vasopressin release into the bloodstream, which in turn increases diuresis resulting in a blood volume reduction that participates in the decrease in BP and/or a decrease in sympathetic tone, decreasing vascular resistance. RB150 constitutes the prototype of a new class of centrally acting antihypertensive agents and is currently being evaluated in a Phase Ib clinical trial.

SDHD immunohistochemistry: a new tool to validate SDHx mutations in pheochromocytoma/paraganglioma.

CONTEXT Pheochromocytomas (PCC) and paragangliomas (PGL) may be caused by a germline mutation in 12 different predisposing genes. We previously reported that immunohistochemistry is a useful approach to detect patients harboring SDHx mutations. SDHA immunostaining is negative in SDHA-mutated tumors only, while SDHB immunostaining is negative in samples mutated on all SDHx genes. In some cases of SDHD or SDHC-mutated tumors, a weak diffuse SDHB labeling has however been described. OBJECTIVE Here, we addressed whether the same procedure could be applicable to detect patients with germline SDHD mutations, by testing two new commercially available anti-SDHD antibodies. DESIGN AND METHODS We performed a retrospective study on 170 PGL/PCC in which we investigated SDHD and SDHB expression by immunohistochemistry. RESULTS SDHx-mutated PGL/PCC showed a completely negative SDHB staining (23/27) or a weak cytoplasmic background (4/27). Unexpectedly, we observed that SDHD immunohistochemistry was positive in SDHx-deficient tumors and negative in the other samples. Twenty-six of 27 SDHx tumors (including the four weakly stained for SDHB) were positive for SDHD. Among non-SDHx tumors, 138/143 were positive for SDHB and negative for SDHD. Five cases showed a negative immunostaining for SDHB, but were negative for SDHD. CONCLUSION Our results demonstrate that a positive SDHD immunostaining predicts the presence of an SDHx gene mutation. Because SDHB negative immunostaining is sometimes difficult to interpret in the case of background, the addition of SDHD positive immunohistochemistry will be a very useful tool to predict or validate SDHx gene variants in PGL/PCC.

New structural insights into the apelin receptor: identification of key residues for apelin binding

Apelin is the endogenous ligand of the orphan 7‐transmembrane domain GPCRAPJ, now named the apelin receptor (ApelinR). Apelin plays a prominent role in body fluid and cardiovascular homeostasis. To better understand the structural organization of the ApelinR, we built 3 homology 3‐dimensional (3D) models of the human ApelinR using the validated cholecystokinin receptor‐1 3D model or the X‐ray structures of the β2‐adrenergic and CXCR4 receptors as templates. Docking of the pyroglutamyl form of apelin 13 (pE13F) into these models revealed the conservation at the bottom of the binding site of a hydrophobic cavity in which the C‐terminal Phe of pE13F was embedded. In contrast, at the top of the binding site, depending on the model, different interactions were visualized between acidic residues of the ApelinR and the basic residues of pE13F. Using site‐directed mutagenesis, we showed that Asp 92, Glu 172, and Asp 282 of rat ApelinR are key residues in apelin binding by interacting with Lys 8, Arg 2, and Arg 4 of pE13F, respectively. These residues are only seen in the CXCR4‐based ApelinR 3D model, further validating this model. These findings bring new insights into the structural organization of the ApelinR and the mode of apelin binding.—Gerbier, R., Leroux, V., Couvineau, P., Alvear‐Perez, R., Maigret, B., Llorens‐Cortes, C., Iturrioz, X., New structural insights into the apelin receptor: identification of key residues for apelin binding. FASEB J. 29, 314–322 (2015). www.fasebj.org

Structure-activity relationship studies toward the discovery of selective apelin receptor agonists.

Apelin is the endogenous ligand for the previously orphaned G protein-coupled receptor APJ. Apelin and its receptor are widely distributed in the brain, heart, and vasculature, and are emerging as an important regulator of body fluid homeostasis and cardiovascular functions. To further progress in the pharmacology and the physiological role of the apelin receptor, the development of small, bioavailable agonists and antagonists of the apelin receptor, is crucial. In this context, E339-3D6 (1) was described as the first nonpeptidic apelin receptor agonist. We show here that 1 is actually a mixture of polymethylated species, and we describe an alternative and versatile solid-phase approach that allows access to highly pure 27, the major component of 1. This approach was also applied to prepare a series of derivatives in order to identify the crucial structural determinants required for the ligand to maintain its affinity for the apelin receptor as well as its capacity to promote apelin receptor signaling and internalization. The study of the structure-activity relationships led to the identification of ligands 19, 21, and 38, which display an increased affinity compared to that of 27. The latter and 19 behave as full agonists with regard to cAMP production and apelin receptor internalization, whereas 21 is a biased agonist toward cAMP production. Interestingly, the three ligands display a much higher stability in mouse plasma (T1/2 > 10 h) than the endogenous apelin-17 peptide 2 (T1/2 < 4 min).