László Szidonya

Dimerization and oligomerization of G-protein-coupled receptors: debated structures with established and emerging functions.

Dimerization or oligomerization of G-protein-coupled receptors (GPCRs) is a novel concept, which may lead to the reevaluation of the actions of pharmacological ligands, hormones, neurotransmitters, and other mediators acting on GPCRs. Although a large number of data obtained using different biophysical, biochemical and structural methods, and functional approaches argue for dimerization or oligomerization of these receptors, several publications criticized the applied methods and challenged the concept. The aim of this paper is to review the data that support the concept of receptor oligomerization, and the most important arguments against it. We conclude that it will require major methodical improvements to obtain decisive proof, whether GPCRs exist in their native membrane environments as homo- or heterodimeric or oligomeric complexes, in which receptor monomers have stable direct interactions. However, overwhelming amounts of data suggest that many GPCRs exhibit functional properties that require direct or indirect interactions between clustered receptors. Although it is difficult to conclude, about the exact nature of these interactions, dimerization or oligomerization of GPCRs is a useful paradigm for pharmacologists to study properties of receptors, which require functionally important clustering of receptors, such as trafficking of newly synthesized receptors to the cell surface, allosteric modulation of ligand binding, signaling specificity, co-internalization, or cross-inhibition of GPCRs.

The Role of Diacylglycerol Lipase in Constitutive and Angiotensin AT 1 Receptor- stimulated Cannabinoid CB1 Receptor Activity *

The cannabinoid CB1 receptor (CB1R) is a G protein-coupled receptor, which couples to the Gi/o family of heterotrimeric G proteins. The receptor displays both basal and agonist-induced signaling and internalization. Although basal activity of CB1Rs is attributed to constitutive (agonist-independent) receptor activity, studies in neurons suggested a role of postsynaptic endocannabinoid (eCB) release in the persistent activity of presynaptic CB1Rs. To elucidate the role of eCBs in basal CB1R activity, we have investigated the role of diacylglycerol lipase (DAGL) in this process in Chinese hamster ovary (CHO) cells, which are not targeted specifically with eCBs. Agonist-induced G protein activation was determined by detecting dissociation G protein subunits expressed in CHO cells with bioluminescence resonance energy transfer (BRET), after labeling the α and β subunits with Renilla luciferase and enhanced yellow fluorescent protein (EYFP), respectively. Preincubation of the cells with tetrahydrolipstatin (THL), a known inhibitor of DAGLs, caused inhibition of the basal activity of CB1R. Moreover, preincubation of CHO and cultured hippocampal neurons with THL increased the number of CB1Rs on the cell membrane, which reflects its inhibitory action on CB1R internalization in non-simulated cells. In CHO cells co-expressing CB1R and angiotensin AT1 receptors, angiotensin II-induced Go protein activation that was blocked by both a CB1R antagonist and THL. These data indicate that cell-derived eCB mediators have a general role in the basal activity of CB1Rs in non-neural cells and neurons, and that this mechanism can be stimulated by AT1 receptor activation.

Paracrine transactivation of the CB1 cannabinoid receptor by AT1 angiotensin and other Gq/11 protein-coupled receptors.

Intracellular signaling systems of G protein-coupled receptors are well established, but their role in paracrine regulation of adjacent cells is generally considered as a tissue-specific mechanism. We have shown previously that AT1 receptor (AT1R) stimulation leads to diacylglycerol lipase-mediated transactivation of co-expressed CB1Rs in Chinese hamster ovary cells. In the present study we detected a paracrine effect of the endocannabinoid release from Chinese hamster ovary, COS7, and HEK293 cells during the stimulation of AT1 angiotensin receptors by determining CB1 cannabinoid receptor activity with bioluminescence resonance energy transfer-based sensors of G protein activation expressed in separate cells. The angiotensin II-induced, paracrine activation of CB1 receptors was visualized by detecting translocation of green fluorescent protein-tagged β-arrestin2. Mass spectrometry analyses have demonstrated angiotensin II-induced stimulation of 2-arachidonoylglycerol production, whereas no increase of anandamide levels was observed. Stimulation of Gq/11-coupled M1, M3, M5 muscarinic, V1 vasopressin, α1a adrenergic, B2 bradykinin receptors, but not Gi/o-coupled M2 and M4 muscarinic receptors, also led to paracrine transactivation of CB1 receptors. These data suggest that, in addition to their retrograde neurotransmitter role, endocannabinoids have much broader paracrine mediator functions during activation of Gq/11-coupled receptors.

Sec14 Homology Domain Targets p50RhoGAP to Endosomes and Provides a Link between Rab and Rho GTPases

Sec14 protein was first identified in Saccharomyces cerevisiae, where it serves as a phosphatidylinositol transfer protein that is essential for the transport of secretory proteins from the Golgi complex. A protein domain homologous to Sec14 was identified in several mammalian proteins that regulates Rho GTPases, including exchange factors and GTPase activating proteins. P50RhoGAP, the first identified GTPase activating protein for Rho GTPases, is composed of a Sec14-like domain and a Rho-GTPase activating protein (GAP) domain. The biological function of its Sec14-like domain is still unknown. Here we show that p50RhoGAP is present on endosomal membranes, where it colocalizes with internalized transferrin receptor. We demonstrate that the Sec14-like domain of P50RhoGAP is responsible for the endosomal targeting of the protein. We also show that overexpression of p50RhoGAP or its Sec14-like domain inhibits transferrin uptake. Furthermore, both P50RhoGAP and its Sec14-like domain show colocalization with small GTPases Rab11 and Rab5. We measured bioluminescence resonance energy transfer between p50RhoGAP and Rab11, indicating that these proteins form molecular complex in vivo on endosomal membranes. The interaction was mediated by the Sec 14-like domain of p50RhoGAP. Our results indicate that Sec14-like domain, which was previously considered as a phospholipid binding module, may have a role in the mediation of protein-protein interactions. We suggest that p50RhoGAP provides a link between Rab and Rho GTPases in the regulation of receptor-mediated endocytosis.

Differential β‐arrestin binding of AT1 and AT2 angiotensin receptors

Agonist stimulation of G protein‐coupled receptors causes receptor activation, phosphorylation, β‐arrestin binding and receptor internalization. Angiotensin II (AngII) causes rapid internalization of the AT1 receptors, whereas AngII‐bound AT2 receptors do not internalize. Although the activation of the rat AT1A receptor with AngII causes translocation of β‐arrestin2 to the receptor, no association of this molecule with the AT2 receptor can be detected after AngII treatment with confocal microscopy or bioluminescence resonance energy transfer. These data demonstrate that the two subtypes of angiotensin receptors have different mechanisms of regulation.

Bioluminescence Resonance Energy Transfer Reveals the Adrenocorticotropin (ACTH)-Induced Conformational Change of the Activated ACTH Receptor Complex in Living Cells

Bioluminescence resonance energy transfer analysis is used to study the interaction between melanocortin 2 receptor (MC2R) accessory protein and the MC2R and provides evidence for protein kinase A-dependent conformational changes in the receptor complex following receptor activation.

Allosteric interactions within the AT1 angiotensin receptor homodimer: Role of the conserved DRY motif

G protein coupled receptor (GPCR) dimerization has a remarkable impact on the diversity of receptor signaling. Allosteric communication between the protomers of the dimer can alter ligand binding, receptor conformation and interactions with different effector proteins. In this study we investigated the allosteric interactions between wild type and mutant protomers of type 1 angiotensin receptor (AT₁R) dimers transiently expressed in CHO cells. In our experimental setup, one protomer of the dimer was selectively stimulated and the β-arrestin2 binding and conformation alteration of the other protomer was followed. The interaction between β-arrestin2 and the non-stimulated protomer was monitored through a bioluminescence resonance energy transfer (BRET) based method. To measure the conformational alterations in the non-stimulated protomer directly, we also used a BRET based intramolecular receptor biosensor, which was created by inserting yellow fluorescent protein (YFP) into the 3rd intracellular loop of AT₁R and fusing Renilla luciferase (RLuc) to its C terminal region. We have detected β-arrestin2 binding, and altered conformation of the non-stimulated protomer. The cooperative ligand binding of the receptor homodimer was also observed by radioligand dissociation experiments. Mutation of the conserved DRY sequence in the activated protomer, which is also required for G protein activation, abolished all the observed allosteric effects. These data suggest that allosteric interactions in the homodimers of AT₁R significantly affect the function of the non-stimulated protomer, and the conserved DRY motif has a crucial role in these interactions.

Angiotensin II-Induced Expression of Brain-Derived Neurotrophic Factor in Human and Rat Adrenocortical Cells

Angiotensin II (Ang II) is a major regulator of steroidogenesis in adrenocortical cells, and is also an effective inducer of cytokine and growth factor synthesis in several cell types. In microarray analysis of H295R human adrenocortical cells, the mRNA of brain-derived neurotrophic factor (BDNF), a neurotrophin widely expressed in the nervous system, was one of the most up-regulated genes by Ang II. The aim of the present study was the analysis of the Ang II-induced BDNF expression and BDNF-induced effects in adrenocortical cells. Real-time PCR studies have shown that BDNF is expressed in H295R and rat adrenal glomerulosa cells. In H295R cells, the kinetics of Ang II-induced BDNF expression was faster than that of aldosterone synthase (CYP11B2). Inhibition of calmodulin kinase by KN93 did not significantly affect the Ang II-induced stimulation of BDNF expression, suggesting that it occurs by a different mechanism from the CYP11B2-response. Ang II also caused candesartan-sensitive, type-1 Ang II receptor-mediated stimulation of BDNF gene expression in primary rat glomerulosa cells. In rat adrenal cortex, BDNF protein was localized to the subcapsular region. Ang II increased BDNF protein levels both in human and rat cells, and BDNF secretion of H295R cells. Ang II also increased type-1 Ang II receptor-mediated BDNF expression in vivo in furosemide-treated rats. In rat glomerulosa cells, BDNF induced tropomyosin-related kinase B receptor-mediated stimulation of EGR1 and TrkB expression. These data demonstrate that Ang II stimulates BDNF expression in human and rat adrenocortical cells, and BDNF may have a local regulatory function in adrenal glomerulosa cells.

Cross-inhibition of angiotensin AT1 receptors supports the concept of receptor oligomerization

G protein-coupled receptors are cell surface receptors that mediate the effects of extracellular signals in the endocrine/paracrine and sensory systems. Experimental evidence is accumulating, which suggest that these receptors form dimers or higher order oligomers. The functional relevance of G protein-coupled receptor dimerization or oligomerization has been raised in a number of different processes, including ontogeny, internalization, ligand-induced regulation, pharmacological diversity and signal transduction of these receptors. Agonist-independent homo- and hetero-oligomerization of the angiotensin AT1 receptor has been reported, and it has been suggested that hetero-oligomerization with beta-adrenergic receptors leads to cross-inhibition of these receptors. Much less is known about the functional interactions between AT1 receptor homo-oligomers. The aim of the present study was to analyze the functional interactions between these homo-oligomers by determining the functions of normal, AT1 receptor blocker (candesartan) resistant (S109Y) and G protein coupling deficient (DRY/AAY) AT1 receptors (co-)expressed in COS-7 cells. Although we have found no evidence that stimulation of a G protein coupling deficient receptor could cross-activate co-expressed normal receptors, candesartan binding to a signaling deficient receptor caused cross-inhibition of co-expressed candesartan resistant AT1 receptors. Since the studied mutations were in the third intracellular helix of the receptor, the observed effects cannot be explained with domain swapping. These data suggest that AT1 receptor blockers cause cross-inhibition of homo-oligomerized AT1 receptors, and support the concept that receptor dimers/oligomers serve as the functional unit of G protein-coupled receptors.

A transgenic model of visceral obesity and the metabolic syndrome

Obesity is often associated with the metabolic syndrome, which includes insulin resistance, glucose intolerance and dyslipidemia. The best predictor of these morbidities is not the total body adipose mass, but the specific quantity of visceral fat, with ‘apple-shaped’ people who carry excess weight around their waists being more susceptible to these illnesses than people who are equally overweight but with the weight around their hips. The role of glucocorticoids in this process remained elusive for many years, because in most obese patients plasma glucocorticoid levels are normal. However, the activity of 11β hydroxysteroid dehydrogenase type 1 enzyme (11β HSD-1), which regenerates active cortisol from inactive cortisone, was recently shown to be greater in abdominal fat than in subcutaneous abdominal tissue, suggesting a role for increased local cortisol activity in human obesity.To test the role of 11β HSD-1 and local glucocorticoid production in the development of visceral obesity and the metabolic syndrome, Masuzaki et al. [1xA transgenic model of visceral obesity and the metabolic syndrome. Masuzaki, H. et al. Science. 2001; 294: 2166–2170Crossref | PubMed | Scopus (1268)See all References][1] created transgenic mice selectively overexpressing 11β HSD-1 in the adipose tissue. The overexpression caused a 2.4-fold increase in 11β HSD-1 activity in the adipose tissue of these mice, a magnitude similar to that found in obese humans. Corticosterone levels in the adipose tissue were significantly elevated (by 15–30%), but the serum levels of the hormone were normal, as were several other parameters, including thymic weight, bone mineral density, lean body mass weight and linear growth, which reflect the consequences of circulating glucocorticoids.By 15 weeks of age, the transgenic mice showed increased sensitivity to weight gain compared with normal mice, and the fat accumulation had a prominent abdominal component: even on a low-fat diet, the transgenic mice carried 37.9% of their adipose weight in the abdominal region compared with 27.5% in non-transgenic mice. Food intake was increased by at least 10%, which paralleled the increase in body weight. These data demonstrate that overexpression of 11β HSD-1 in adipose tissue produces disproportionate fat accumulation and hyperphagia.The transgenic mice also seemed to exhibit most symptoms of the metabolic syndrome, including pronounced insulin-resistant diabetes, hyperlipidemia and hyperphagia, in spite of showing hyperleptinemia. The expression of several genes that could influence systemic metabolic pathways was also studied in these mice: mRNA encoding resistin and Acrp30-AdipoQ decreased, whereas angiotensinogen expression in the mesenteric fat increased; mitochondrial uncoupling protein-1 mRNA was significantly decreased in brown adipose tissue.This provides evidence that a modest increase in the activity of 11β HSD-1 in the adipose tissue of mice, similar to that found in obese humans, can cause hyperphagia, visceral obesity and most symptoms of the metabolic syndrome. The authors suggest that this enzyme could be a potential pharmaceutical target for the treatment of this prevalent problem. In fact, it has been shown that thiazolidinedione antidiabetic agents markedly reduce 11β HSD-1 mRNA and activity in adipose tissue. Based on the data of Masuzaki et al., this could be a mechanism for their visceral fat-reducing action in humans, and might play a role in their antidiabetic effects.