Susanna Cotecchia

Constitutively active mutants of the alpha 1B-adrenergic receptor: role of highly conserved polar amino acids in receptor activation.

Site‐directed mutagenesis and molecular dynamics simulations of the alpha 1B‐adrenergic receptor (AR) were combined to explore the potential molecular changes correlated with the transition from R (inactive state) to R (active state). Using molecular dynamics analysis we compared the structural/dynamic features of constitutively active mutants with those of the wild type and of an inactive alpha 1B‐AR to build a theoretical model which defines the essential features of R and R. The results of site‐directed mutagenesis were in striking agreement with the predictions of the model supporting the following hypothesis. (i) The equilibrium between R and R depends on the equilibrium between the deprotonated and protonated forms, respectively, of D142 of the DRY motif. In fact, replacement of D142 with alanine confers high constitutive activity to the alpha 1B‐AR. (ii) The shift of R143 of the DRY sequence out of a conserved ‘polar pocket’ formed by N63, D91, N344 and Y348 is a feature common to all the active structures, suggesting that the role of R143 is fundamental for mediating receptor activation. Disruption of these intramolecular interactions by replacing N63 with alanine constitutively activates the alpha 1B‐AR. Our findings might provide interesting generalities about the activation process of G protein‐coupled receptors.

Oligomerization of the α1a- and α1b-Adrenergic Receptor Subtypes POTENTIAL IMPLICATIONS IN RECEPTOR INTERNALIZATION

We combined biophysical, biochemical, and pharmacological approaches to investigate the ability of the α1a- and α1b-adrenergic receptor (AR) subtypes to form homo- and hetero-oligomers. Receptors tagged with different epitopes (hemagglutinin and Myc) or fluorescent proteins (cyan and green fluorescent proteins) were transiently expressed in HEK-293 cells either individually or in different combinations. Fluorescence resonance energy transfer measurements provided evidence that both the α1a- and α1b-AR can form homo-oligomers with similar transfer efficiency of ∼0.10. Hetero-oligomers could also be observed between the α1b- and the α1a-AR subtypes but not between the α1b-AR and the β2-AR, the NK1 tachykinin, or the CCR5 chemokine receptors. Oligomerization of the α1b-AR did not require the integrity of its C-tail, of two glycophorin motifs, or of the N-linked glycosylation sites at its N terminus. In contrast, helix I and, to a lesser extent, helix VII were found to play a role in the α1b-AR homo-oligomerization. Receptor oligomerization was not influenced by the agonist epinephrine or by the inverse agonist prazosin. A constitutively active (A293E) as well as a signaling-deficient (R143E) mutant displayed oligomerization features similar to those of the wild type α1b-AR. Confocal imaging revealed that oligomerization of the α1-AR subtypes correlated with their ability to co-internalize upon exposure to the agonist. The α1a-selective agonist oxymetazoline induced the co-internalization of the α1a- and α1b-AR, whereas the α1b-AR could not co-internalize with the NK1 tachykinin or CCR5 chemokine receptors. Oligomerization might therefore represent an additional mechanism regulating the physiological responses mediated by the α1a- and α1b-AR subtypes.

Constitutively active G protein-coupled receptors: potential mechanisms of receptor activation.

Mutations of G protein-coupled receptors can increase their constitutive (agonist-independent) activity. Some of these mutations have been artificially introduced by site-directed mutagenesis, others occur spontaneously in human diseases. The analysis of the constitutively active G protein-coupled receptors has provided important informations about the molecular mechanisms underlying receptor activation and drug action.

Anchoring of both PKA and 14‐3‐3 inhibits the Rho‐GEF activity of the AKAP‐Lbc signaling complex

A‐kinase anchoring proteins (AKAPs) target the cAMP‐regulated protein kinase (PKA) to its physiological substrates. We recently identified a novel anchoring protein, called AKAP‐Lbc, which functions as a PKA‐targeting protein as well as a guanine nucleotide exchange factor (GEF) for RhoA. We demonstrated that AKAP‐Lbc Rho‐GEF activity is stimulated by the alpha subunit of the heterotrimeric G protein G12. Here, we identified 14‐3‐3 as a novel regulatory protein interacting with AKAP‐Lbc. Elevation of the cellular concentration of cAMP activates the PKA holoenzyme anchored to AKAP‐Lbc, which phosphorylates the anchoring protein on the serine 1565. This phosphorylation event induces the recruitment of 14‐3‐3, which inhibits the Rho‐GEF activity of AKAP‐Lbc. AKAP‐Lbc mutants that fail to interact with PKA or with 14‐3‐3 show a higher basal Rho‐GEF activity as compared to the wild‐type protein. This suggests that, under basal conditions, 14‐3‐3 maintains AKAP‐Lbc in an inactive state. Therefore, while it is known that AKAP‐Lbc activity can be stimulated by Gα12, in this study we demonstrated that it is inhibited by the anchoring of both PKA and 14‐3‐3.

Characterization of the phosphorylation sites involved in G protein-coupled receptor kinase- and protein kinase C-mediated desensitization of the alpha1B-adrenergic receptor.

Catecholamines as well as phorbol esters can induce the phosphorylation and desensitization of the α1B-adrenergic receptor (α1BAR). In this study, phosphoamino acid analysis of the phosphorylated α1BAR revealed that both epinephrine- and phorbol ester-induced phosphorylation predominantly occurs at serine residues of the receptor. The findings obtained with receptor mutants in which portions of the C-tail were truncated or deleted indicated that a region of 21 amino acids (393–413) of the carboxyl terminus including seven serines contains the main phosphorylation sites involved in agonist- as well as phorbol ester-induced phosphorylation and desensitization of the α1BAR. To identify the serines invoved in agonist- versus phorbol ester-dependent regulation of the receptor, two different strategies were adopted, the seven serines were either substituted with alanine or reintroduced into a mutant lacking all of them. Our findings indicate that Ser394 and Ser400 were phosphorylated following phorbol ester-induced activation of protein kinase C, whereas Ser404, Ser408, and Ser410 were phosphorylated upon stimulation of the α1BAR with epinephrine. The observation that overexpression of G protein-coupled kinase 2 (GRK2) could increase agonist-induced phosphorylation of Ser404, Ser408, and Ser410, strongly suggests that these serines are the phosphorylation sites of the α1BAR for kinases of the GRK family. Phorbol ester-induced phosphorylation of the Ser394 and Ser400 as well as GRK2-mediated phosphorylation of the Ser404, Ser408, and Ser410, resulted in the desensitization of α1BAR-mediated inositol phosphate response. This study provides generalities about the biochemical mechanisms underlying homologous and heterologous desensitization of G protein-coupled receptors linked to the activation of phospholipase C.

The A-kinase anchoring protein (AKAP)-Lbc-signaling complex mediates α1 adrenergic receptor-induced cardiomyocyte hypertrophy

In response to various pathological stresses, the heart undergoes a pathological remodeling process that is associated with cardiomyocyte hypertrophy. Because cardiac hypertrophy can progress to heart failure, a major cause of lethality worldwide, the intracellular signaling pathways that control cardiomyocyte growth have been the subject of intensive investigation. It has been known for more than a decade that the small molecular weight GTPase RhoA is involved in the signaling pathways leading to cardiomyocyte hypertrophy. Although some of the hypertrophic pathways activated by RhoA have now been identified, the identity of the exchange factors that modulate its activity in cardiomyocytes is currently unknown. In this study, we show that AKAP-Lbc, an A-kinase anchoring protein (AKAP) with an intrinsic Rho-specific guanine nucleotide exchange factor activity, is critical for activating RhoA and transducing hypertrophic signals downstream of α1-adrenergic receptors (ARs). In particular, our results indicate that suppression of AKAP-Lbc expression by infecting rat neonatal ventricular cardiomyocytes with lentiviruses encoding AKAP-Lbc-specific short hairpin RNAs strongly reduces both α1-AR-mediated RhoA activation and hypertrophic responses. Interestingly, α1-ARs promote AKAP-Lbc activation via a pathway that requires the α subunit of the heterotrimeric G protein G12. These findings identify AKAP-Lbc as the first Rho-guanine nucleotide exchange factor (GEF) involved in the signaling pathways leading to cardiomyocytes hypertrophy.

Constitutively active mutants of the β1-adrenergic receptor

We provide the first evidence that point mutations can constitutively activate the β1‐adrenergic receptor (AR). Leucine 322 of the β1‐AR in the C‐terminal portion of its third intracellular loop was replaced with seven amino acids (I, T, E, F, C, A and K) differing in their physico‐chemical properties. The β1‐AR mutants expressed in HEK‐293 cells displayed various levels of constitutive activity which could be partially inhibited by some beta‐blockers. The results of this study might have interesting implications for future studies aiming at elucidating the activation process of the β1‐AR as well as the mechanism of action of beta‐blockers.

Selective changes of receptor binding in brain regions of aged rats

Binding to several receptors was compared in brain regions of 3 and 21-23 month-old rats. In crude membrane preparations of aged rats the number of dopamine antagonist receptors in striatum was much reduced (-53%). beta-Noradrenergic receptors (cortex) and benzodiazepine receptors (hippocampus and cerebellum) were less but significantly reduced and serotonergic receptors, alpha 1 noradrenergic receptors (both in cortex) and dopamine agonist receptors (striatum) were unchanged. For each receptor binding the KD values were the same in young and old animals. GABA receptor binding (hippocampus and cerebellum) evaluated at only one 3H-GABA concentration (8 nM) was similar in both groups when expressed per protein content but significantly reduced in aged rats when expressed per tissue wet weight because of the partial purification of the synaptic membranes used for 3H-GABA binding. In our experimental conditions age-related changes of specific binding sites in the central nervous system were selective for some receptors studied and did not seem to be due to general non-specific modification of brain tissue composition.

Dual couplings of the cloned 5-HT1A receptor to both adenylyl cyclase and phospholipase C is mediated via the same Gi protein

The cloned 5-HT1A receptor, stably expressed in HeLa cells, has been shown to mediate the effects of 5-hydroxytryptamine (5-HT) to inhibit cAMP formation and to stimulate the hydrolysis of phosphatidylinositol. Both responses were found to be pertussis toxin sensitive. We have examined these two responses in membranes derived from these cells and show that the 5-HT1A receptor can directly regulate the activity of adenylyl cyclase and phospholipase C in response to agonist. In order to examine whether the same or distinct guanine nucleotide-binding regulatory protein(s) (G protein) are involved in these two signal transduction pathways, we used anti-peptide antibodies recognizing the alpha-subunits of Gi1, Gi2, Gi3 as specific tools, since these pertussis toxin substrates are expressed in HeLa cells. These antibodies have previously been shown to prevent receptor-G protein coupling by binding to the regions of G proteins which are putatively involved in interaction with receptors. Our results indicate that the Gi proteins, but preferentially Gi3, mediate the effects of 5-HT both to inhibit adenylyl cyclase and to stimulate phospholipase C. These findings demonstrate that the same receptor interacting with the same G protein can regulate several distinct effector molecules.

The α1-adrenergic receptors: diversity of signaling networks and regulation

The α1-adrenergic receptor (AR) subtypes (α1a, α1b, and α1d) mediate several physiological effects of epinephrine and norepinephrine. Despite several studies in recombinant systems and insight from genetically modified mice, our understanding of the physiological relevance and specificity of the α1-AR subtypes is still limited. Constitutive activity and receptor oligomerization have emerged as potential features regulating receptor function. Another recent paradigm is that βarrestins and G protein-coupled receptors themselves can act as scaffolds binding a variety of proteins and this can result in growing complexity of the receptor-mediated cellular effects. The aim of this review is to summarize our current knowledge on some recently identified functional paradigms and signaling networks that might help to elucidate the functional diversity of the α1-AR subtypes in various organs.