Susanna Cotecchia - Academia.edu (original) (raw)
Papers by Susanna Cotecchia
Journal of gastrointestinal and liver diseases : JGLD, 2008
Obesity is an excess of fat mass. Fat mass is an energy depot but also an endocrine organ. A dere... more Obesity is an excess of fat mass. Fat mass is an energy depot but also an endocrine organ. A deregulation of the sympathetic nervous system (SNS) might produce obesity. Stress exaggerates diet-induced obesity. After stress, SNS fibers release neuropeptide Y (NPY) which directly increases visceral fat mass producing a metabolic syndrome (MbS)-like phenotype. Adrenergic receptors are the main regulators of lipolysis. In severe obesity, we demonstrated that the adrenergic receptor subtypes are differentially expressed in different fat depots. Liver and visceral fat share a common sympathetic pathway, which might explain the low-grade inflammation which simultaneously occurs in liver and fat of the obese with MbS. The neuroendocrine melanocortinergic system and gastric ghrelin are also greatly deregulated in obesity. A specific mutation in the type 4 melanocortin receptor induces early obesity onset, hyperphagia and insulin-resistance. Nonetheless, it was recently discovered that a muta...
Cellular Signalling, 2015
Cardiac hypertrophy is a complex remodeling process of the heart induced by physiological or path... more Cardiac hypertrophy is a complex remodeling process of the heart induced by physiological or pathological stimuli resulting in increased cardiomyocyte size and myocardial mass. Whereas cardiac hypertrophy can be an adaptive mechanism to stressful conditions of the heart, prolonged hypertrophy can lead to heart failure which represents the primary cause of human morbidity and mortality. Among G protein-coupled receptors, the α 1 -adrenergic receptors (α 1 -ARs) play an important role in the development of cardiac hypertrophy as demonstrated by numerous studies in the past decades, both in primary cardiomyocyte cultures and genetically modified mice. The results of these studies have provided evidence of a large variety of α 1 -AR-induced signaling events contributing to the defining molecular and cellular features of cardiac hypertrophy. Recently, novel signaling mechanisms have been identified and new hypotheses have emerged concerning the functional role of the α 1 -adrenergic receptors in the heart. This review will summarize the main signaling pathways activated by the α 1 -AR in the heart and their functional implications in cardiac hypertrophy.
In the DDTâ MF-2 hamster vas deferens smooth muscle cell line the ..cap alpha..â-adrenergic rec... more In the DDTâ MF-2 hamster vas deferens smooth muscle cell line the ..cap alpha..â-adrenergic receptor (..cap alpha..â-AR) agonist norepinephrine (NE) promotes rapid attenuation of ..cap alpha..â-AR-mediated phosphatidylinositol (PI) metabolism which is paralleled by rapid phosphorylation of the ..cap alpha..â-AR. Cells were labeled by incubation with ³²P/sub i/. Coincubation with NE (100 ..mu..M) significantly increases the rate of ³²P-labeling of both
In this study, we describe a patient with a phenotype of complete hypogonadotropic hypogonadism w... more In this study, we describe a patient with a phenotype of complete hypogonadotropic hypogonadism who presented primary failure of pulsatile GnRH therapy, but responded to exogenous gonadotropin administration. This patient bore a novel point mutation (T for A) at codon 168 of the gene encoding the GnRH receptor (GnRH-R), re- sulting in a serine to arginine change in the fourth
Trends in Pharmacological Sciences, 1993
Adrenoceptors are prototypic members of the superfamily of seven transmembrane domain, G protein-... more Adrenoceptors are prototypic members of the superfamily of seven transmembrane domain, G protein-coupled receptors. Study of the properties of several mutationally activated adrenoceptors is deepening understanding of the normal functioning of this ubiquitous class of receptors. The new findings suggest an expansion of the classical ternary complex model of receptor action to include an explicit isomerization of the receptors from an inactive to an active state which couples to the G protein ('allosteric ternary complex model'). This isomerization involves conformational changes which may occur spontaneously, or be induced by agonists or appropriate mutations which abrogate the normal 'constraining' function of the receptor, allowing it to 'relax' into the active conformation. Robert Lefkowitz and colleagues discuss the physiological and pathophysiological implications of these new insights into regulation of receptor activity.
Trends in Pharmacological Sciences, 2005
Science, 1993
The actions of many hormones and neurotransmitters are mediated by the members of a superfamily o... more The actions of many hormones and neurotransmitters are mediated by the members of a superfamily of receptors coupled to heterotrimeric guanine nucleotide-binding proteins (G proteins). These receptors are characterized by a highly conserved topographical arrangement in which seven transmembrane domains are connected by intracellular and extracellular loops. The interaction between these receptors and G proteins is mediated in large part by the third intracellular loop of the receptor. Coexpression of the third intracellular loop of the alpha 1B-adrenergic receptor with its parent receptor inhibited receptor-mediated activation of phospholipase C. The inhibition extended to the closely related alpha 1C-adrenergic receptor subtype, but not the phospholipase C-coupled M1 muscarinic acetylcholine receptor nor the adenylate cyclase-coupled D1A dopamine receptor. These results suggest that the receptor-G protein interface may represent a target for receptor antagonist drugs.
Proteins: Structure, Function, and Genetics, 1999
This study proposes a theoretical model describing the electrostatically driven step of the ␣1b-a... more This study proposes a theoretical model describing the electrostatically driven step of the ␣1b-adrenergic receptor (AR)-G protein recognition. The comparative analysis of the structuraldynamics features of functionally different receptor forms, i.e., the wild type (ground state) and its constitutively active mutants D142A and A293E, was instrumental to gain insight on the receptor-G protein electrostatic and steric complementarity. Rigid body docking simulations between the different forms of the ␣1b-AR and the heterotrimeric G␣q, G␣s, G␣i1, and G␣t suggest that the cytosolic crevice shared by the active receptor and including the second and the third intracellular loops as well as the cytosolic extension of helices 5 and 6, represents the receptor surface with docking complementarity with the G protein. On the other hand, the G protein solvent-exposed portions that recognize the intracellular loops of the activated receptors are the Nterminal portion of ␣3, ␣G, the ␣G/␣4 loop, ␣4, the ␣4/6 loop, ␣5, and the C-terminus. Docking simulations suggest that the two constitutively active mutants D142A and A293E recognize different G proteins with similar selectivity orders, i.e., G␣q Ա G␣s Ͼ G␣i Ͼ Ͼ G␣t. The theoretical models herein proposed might provide useful suggestions for new experiments aiming at exploring the receptor-G protein interface. Proteins 1999;37:145-156.
Proceedings of the National Academy of Sciences, 1990
The adrenergic receptors (ARs) (subtypes alpha 1, alpha 2, beta 1, and beta 2) are a prototypic f... more The adrenergic receptors (ARs) (subtypes alpha 1, alpha 2, beta 1, and beta 2) are a prototypic family of guanine nucleotide binding regulatory protein-coupled receptors that mediate the physiological effects of the hormone epinephrine and the neurotransmitter norepinephrine. We have previously assigned the genes for beta 2- and alpha 2-AR to human chromosomes 5 and 10, respectively. By Southern analysis of somatic cell hybrids and in situ chromosomal hybridization, we have now mapped the alpha 1-AR gene to chromosome 5q32----q34, the same position as beta 2-AR, and the beta 1-AR gene to chromosome 10q24----q26, the region where alpha 2-AR is located. In mouse, both alpha 2- and beta 1-AR genes were assigned to chromosome 19, and the alpha 1-AR locus was localized to chromosome 11. Pulsed field gel electrophoresis has shown that the alpha 1- and beta 2-AR genes in humans are within 300 kilobases (kb) and the distance between the alpha 2- and beta 1-AR genes is less than 225 kb. The proximity of these two pairs of AR genes and the sequence similarity that exists among all the ARs strongly suggest that they are evolutionarily related. Moreover, they likely arose from a common ancestral receptor gene and subsequently diverged through gene duplication and chromosomal duplication to perform their distinctive roles in mediating the physiological effects of catecholamines. The AR genes thus provide a paradigm for understanding the evolution of such structurally conserved yet functionally divergent families of receptor molecules.
Proceedings of the National Academy of Sciences, 1997
To investigate the functional role of different ␣ 1 -adrenergic receptor (␣ 1 -AR) subtypes in vi... more To investigate the functional role of different ␣ 1 -adrenergic receptor (␣ 1 -AR) subtypes in vivo, we have applied a gene targeting approach to create a mouse model lacking the ␣ 1b -AR (␣ 1b ؊͞؊). Reverse transcription-PCR and ligand binding studies were combined to elucidate the expression of the ␣ 1 -AR subtypes in various tissues of ␣ 1b ؉͞؉ and ؊͞؊ mice. Total ␣ 1 -AR sites were decreased by 98% in liver, 74% in heart, and 42% in cerebral cortex of the ␣ 1b ؊͞؊ as compared with ؉͞؉ mice. Because of the large decrease of ␣ 1 -AR in the heart and the loss of the ␣ 1b -AR mRNA in the aorta of the ␣ 1b ؊͞؊ mice, the in vivo blood pressure and in vitro aorta contractile responses to ␣ 1 -agonists were investigated in ␣ 1b ؉͞؉ and ؊͞؊ mice. Our findings provide strong evidence that the ␣ 1b -AR is a mediator of the blood pressure and the aorta contractile responses induced by ␣ 1 agonists. This was demonstrated by the finding that the mean arterial blood pressure response to phenylephrine was decreased by 45% in ␣ 1b ؊͞؊ as compared with ؉͞؉ mice. In addition, phenylephrine-induced contractions of aortic rings also were decreased by 25% in ␣ 1b ؊͞؊ mice. The ␣ 1b -AR knockout mouse model provides a potentially useful tool to elucidate the functional specificity of different ␣ 1 -AR subtypes, to better understand the effects of adrenergic drugs, and to investigate the multiple mechanisms involved in the control of blood pressure.
Proceedings of the National Academy of Sciences, 2007
In response to various pathological stresses, the heart undergoes a pathological remodeling proce... more 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 ␣1-adrenergic receptors (ARs). In particular, our results indicate that suppression of AKAP-Lbc expression by infecting rat neonatal ventricular cardiomyocytes with lentiviruses encoding AKAP-Lbcspecific short hairpin RNAs strongly reduces both ␣1-AR-mediated RhoA activation and hypertrophic responses. Interestingly, ␣1-ARs promote AKAP-Lbc activation via a pathway that requires the ␣ 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.
Proceedings of the National Academy of Sciences, 1997
In this study, a quantitative approach was used to investigate the role of D142, which belongs to... more In this study, a quantitative approach was used to investigate the role of D142, which belongs to the highly conserved E͞DRY sequence, in the activation process of the ␣ 1B -adrenergic receptor (␣ 1B -AR). Experimental and computer-simulated mutagenesis were performed by substituting all possible natural amino acids at the D142 site. The resulting congeneric set of proteins together with the finding that all the receptor mutants show various levels of constitutive (agonist-independent) activity enabled us to quantitatively analyze the relationships between structural͞dynamic features and the extent of constitutive activity. Our results suggest that the hydrophobic͞hydrophilic character of D142, which could be regulated by protonation͞deprotonation of this residue, is an important modulator of the transition between the inactive (R) and active (R*) state of the ␣ 1B -AR. Our study represents an example of quantitative structureactivity relationship analysis of the activation process of a G protein-coupled receptor.
Tumor promoting phorbol esters stimulate Ca/sup + +/ phospholipid-dependent protein kinase C. It ... more Tumor promoting phorbol esters stimulate Ca/sup + +/ phospholipid-dependent protein kinase C. It has been suggested that this enzyme regulates the functional properties of different cell membrane receptors. In this study we investigated the effect of phorbol esters on ..cap alpha..â-adrenoceptor binding and phosphatidylinositol metabolism in cultured smooth muscle cells derived from rabbit aorta. Treatment of these cells with biologically active phorbol esters for 15 min. to 2 hours caused a marked decrease of norepinephrine stimulation of inositol phospholipid metabolism and a 3 fold decrease in agonist affinity for ¹²âµI-HEAT binding to ..cap alpha..â-adrenoceptors in the intact smooth muscle cells. The ability of phorbol esters to modulate ..cap alpha..â-adrenoceptor responsiveness suggests that activation of protein kinase C may represent an important mechanism regulating ..cap alpha..â-adrenergic receptor functional properties. 32 references, 4 figures.
Obesity, 2007
GARRUTI, GABRIELLA, VITTORIO GIUSTI, JÜ RG NUSSBERGER, CHRISTIAN DARIMONT, CHANTAL VERDUMO, CATHE... more GARRUTI, GABRIELLA, VITTORIO GIUSTI, JÜ RG NUSSBERGER, CHRISTIAN DARIMONT, CHANTAL VERDUMO, CATHERINE AMSTUTZ, FRANCESCO PUGLISI, FRANCESCO GIORGINO, RICCARDO GIORGINO, AND SUSANNA COTECCHIA. Expression and secretion of the atrial natriuretic peptide in human adipose tissue and preadipocytes. Obesity. 2007;15: 2181-2189.
Neurobiology of Learning and Memory, 2001
Knockout mice lacking the alpha-1b adrenergic receptor were tested in behavioral experiments. Rea... more Knockout mice lacking the alpha-1b adrenergic receptor were tested in behavioral experiments. Reaction to novelty was first assessed in a simple test in which the time taken by the knockout mice and their littermate controls to enter a second compartment was compared. Then the mice were tested in an open field to which unknown objects were subsequently added. Special novelty was introduced by moving one of the familiar objects to another location in the open field. Spatial behavior and memory were further studied in a homing board test, and in the water maze. The alpha-1b knockout mice showed an enhanced reactivity to new situations. They were faster to enter the new environment, covered longer paths in the open field, and spent more time exploring the new objects. They reacted like controls to modification inducing spatial novelty. In the homing board test, both the knockout mice and the control mice seemed to use a combination of distant visual and proximal olfactory cues, showing place preference only if the two types of cues were redundant. In the water maze the alpha-1b knockout mice were unable to learn the task, which was confirmed in a probe trial without platform. They were perfectly able, however, to escape in a visible platform procedure. These results confirm previous findings showing that the noradrenergic pathway is important for the modulation of behaviors such as reaction to novelty and exploration, and suggest that this is mediated, at least partly, through the alpha-1b adrenergic receptors. The lack of alpha-1b adrenergic receptors in spatial orientation does not seem important in cue-rich tasks but may interfere with orientation in situations providing distant cues only. ᭧
Naunyn-Schmiedeberg's Archives of Pharmacology, 2001
In order to characterize inverse agonism at α 1Badrenoceptors, we have compared the concentration... more In order to characterize inverse agonism at α 1Badrenoceptors, we have compared the concentration-response relationships of several quinazoline and non-quinazoline α 1 -adrenoceptor antagonists at cloned hamster wildtype (WT) α 1B -adrenoceptors and a constitutively active mutant (CAM) thereof upon stable expression in Rat-1 fibroblasts. Receptor activation or inhibition thereof was assessed as [ 3 H]inositol phosphate (IP) accumulation. Quinazoline (alfuzosin, doxazosin, prazosin, terazosin) and non-quinazoline α 1 -adrenoceptor antagonists (BE 2254, SB 216,469, tamsulosin) concentration-dependently inhibited phenylephrine-stimulated IP formation at both WT and CAM with K i values similar to those previously found in radioligand binding studies. At CAM in the absence of phenylephrine, the quinazolines produced concentrationdependent inhibition of basal IP formation; the maximum inhibition was ~55%, and the corresponding EC 50 values were slightly smaller than the K i values. In contrast, BE 2254 produced much less inhibition of basal IP formation, SB 216,469 was close to being a neutral antagonist, and tamsulosin even weakly stimulated IP formation. The inhibitory effects of the quinazolines and BE 2254 as well as the stimulatory effect of tamsulosin were equally blocked by SB 216,469 at CAM. At WT in the absence of phenylephrine, tamsulosin did not cause significant stimulation and none of the other compounds caused significant inhibition of basal IP formation. We conclude that α 1 -adrenoceptor antagonsits with a quinazoline structure exhibit greater efficacy as inverse agonists than those without.
Methods, 1998
Acting either coordinately or independently, these two This work describes the ab initio procedur... more Acting either coordinately or independently, these two This work describes the ab initio procedure employed to build species bind and modulate the activities of downstream an activation model for the a 1b -adrenergic receptor (a 1b -AR). The effector molecules. G proteins are released from effirst version of the model was progressively modified and complifectors on the breakdown of GTP that results from the cated by means of a many-step iterative procedure characterized slow GTPase activity of the a subunit. The inactive a by the employment of experimental validations of the model in subunit can recombine with bg, reforming the heteroeach upgrading step. A combined simulated (molecular dynamtrimer, which can then reassociate with its receptor ics) and experimental mutagenesis approach was used to deterand undergo a new cycle of signal transduction (3). mine the structural and dynamic features characterizing the in-Despite the fact that recent crystallographic studies active and active states of a 1b -AR. The latest version of the of G protein a subunits and heterotrimers have begun Recently, biophysical and biochemical studies on rhodopsin showed that a relative displacement of helices
Life Sciences, 2009
Aim: Alpha1-adrenergic receptors (α 1 -ARs) are classified into three subtypes: α 1A -AR, α 1B -A... more Aim: Alpha1-adrenergic receptors (α 1 -ARs) are classified into three subtypes: α 1A -AR, α 1B -AR, and α 1D -AR. Triple disruption of α 1A -AR, α 1B -AR, and α 1D -AR genes results in hypotension and produces no contractile response of the thoracic aorta to noradrenalin. Presently, we characterized vascular contractility against other vasoconstrictors, such as potassium, prostaglandin F2alpha (PGF 2α ) and 5-hydroxytryptamine (5-HT), in α 1A -AR, α 1B -AR, and α 1D -AR triple knockout (α 1 -AR triple KO) mice. Main methods: The contractile responses to the stimulation with vasoconstrictors were studied using isolated thoracic aorta. Key findings: As a result, the phasic and tonic contraction induced by a high concentration of potassium (20 mM) was enhanced in the isolated thoracic aorta of α 1 -AR triple KO mice compared with that of wild-type (WT) mice. In addition, vascular responses to PGF 2α and 5-HT were also enhanced in the isolated thoracic aorta of α 1 -AR triple KO mice compared with WT mice. Similar to in vitro findings with isolated thoracic aorta, in vivo pressor responses to PGF 2α were enhanced in α 1 -AR triple KO mice. Real-time reverse transcription-polymerase chain reaction analysis and western blot analysis indicate that gene expression of the 5-hydroxytryptamine 2A (5-HT 2A ) receptor was up-regulated in the thoracic aorta of α 1 -AR triple KO mice while the prostaglandin F2α receptor (FP) was unchanged. Significance: These results suggest that loss of α 1 -ARs can lead to enhancement of vascular responsiveness to the vasoconstrictors and may imply that α 1 -ARs and the subsequent signaling regulate the vascular responsiveness to other stimulations such as depolarization, 5-HT and PGF 2α .
Journal of Surgical Research, 1991
Journal of gastrointestinal and liver diseases : JGLD, 2008
Obesity is an excess of fat mass. Fat mass is an energy depot but also an endocrine organ. A dere... more Obesity is an excess of fat mass. Fat mass is an energy depot but also an endocrine organ. A deregulation of the sympathetic nervous system (SNS) might produce obesity. Stress exaggerates diet-induced obesity. After stress, SNS fibers release neuropeptide Y (NPY) which directly increases visceral fat mass producing a metabolic syndrome (MbS)-like phenotype. Adrenergic receptors are the main regulators of lipolysis. In severe obesity, we demonstrated that the adrenergic receptor subtypes are differentially expressed in different fat depots. Liver and visceral fat share a common sympathetic pathway, which might explain the low-grade inflammation which simultaneously occurs in liver and fat of the obese with MbS. The neuroendocrine melanocortinergic system and gastric ghrelin are also greatly deregulated in obesity. A specific mutation in the type 4 melanocortin receptor induces early obesity onset, hyperphagia and insulin-resistance. Nonetheless, it was recently discovered that a muta...
Cellular Signalling, 2015
Cardiac hypertrophy is a complex remodeling process of the heart induced by physiological or path... more Cardiac hypertrophy is a complex remodeling process of the heart induced by physiological or pathological stimuli resulting in increased cardiomyocyte size and myocardial mass. Whereas cardiac hypertrophy can be an adaptive mechanism to stressful conditions of the heart, prolonged hypertrophy can lead to heart failure which represents the primary cause of human morbidity and mortality. Among G protein-coupled receptors, the α 1 -adrenergic receptors (α 1 -ARs) play an important role in the development of cardiac hypertrophy as demonstrated by numerous studies in the past decades, both in primary cardiomyocyte cultures and genetically modified mice. The results of these studies have provided evidence of a large variety of α 1 -AR-induced signaling events contributing to the defining molecular and cellular features of cardiac hypertrophy. Recently, novel signaling mechanisms have been identified and new hypotheses have emerged concerning the functional role of the α 1 -adrenergic receptors in the heart. This review will summarize the main signaling pathways activated by the α 1 -AR in the heart and their functional implications in cardiac hypertrophy.
In the DDTâ MF-2 hamster vas deferens smooth muscle cell line the ..cap alpha..â-adrenergic rec... more In the DDTâ MF-2 hamster vas deferens smooth muscle cell line the ..cap alpha..â-adrenergic receptor (..cap alpha..â-AR) agonist norepinephrine (NE) promotes rapid attenuation of ..cap alpha..â-AR-mediated phosphatidylinositol (PI) metabolism which is paralleled by rapid phosphorylation of the ..cap alpha..â-AR. Cells were labeled by incubation with ³²P/sub i/. Coincubation with NE (100 ..mu..M) significantly increases the rate of ³²P-labeling of both
In this study, we describe a patient with a phenotype of complete hypogonadotropic hypogonadism w... more In this study, we describe a patient with a phenotype of complete hypogonadotropic hypogonadism who presented primary failure of pulsatile GnRH therapy, but responded to exogenous gonadotropin administration. This patient bore a novel point mutation (T for A) at codon 168 of the gene encoding the GnRH receptor (GnRH-R), re- sulting in a serine to arginine change in the fourth
Trends in Pharmacological Sciences, 1993
Adrenoceptors are prototypic members of the superfamily of seven transmembrane domain, G protein-... more Adrenoceptors are prototypic members of the superfamily of seven transmembrane domain, G protein-coupled receptors. Study of the properties of several mutationally activated adrenoceptors is deepening understanding of the normal functioning of this ubiquitous class of receptors. The new findings suggest an expansion of the classical ternary complex model of receptor action to include an explicit isomerization of the receptors from an inactive to an active state which couples to the G protein ('allosteric ternary complex model'). This isomerization involves conformational changes which may occur spontaneously, or be induced by agonists or appropriate mutations which abrogate the normal 'constraining' function of the receptor, allowing it to 'relax' into the active conformation. Robert Lefkowitz and colleagues discuss the physiological and pathophysiological implications of these new insights into regulation of receptor activity.
Trends in Pharmacological Sciences, 2005
Science, 1993
The actions of many hormones and neurotransmitters are mediated by the members of a superfamily o... more The actions of many hormones and neurotransmitters are mediated by the members of a superfamily of receptors coupled to heterotrimeric guanine nucleotide-binding proteins (G proteins). These receptors are characterized by a highly conserved topographical arrangement in which seven transmembrane domains are connected by intracellular and extracellular loops. The interaction between these receptors and G proteins is mediated in large part by the third intracellular loop of the receptor. Coexpression of the third intracellular loop of the alpha 1B-adrenergic receptor with its parent receptor inhibited receptor-mediated activation of phospholipase C. The inhibition extended to the closely related alpha 1C-adrenergic receptor subtype, but not the phospholipase C-coupled M1 muscarinic acetylcholine receptor nor the adenylate cyclase-coupled D1A dopamine receptor. These results suggest that the receptor-G protein interface may represent a target for receptor antagonist drugs.
Proteins: Structure, Function, and Genetics, 1999
This study proposes a theoretical model describing the electrostatically driven step of the ␣1b-a... more This study proposes a theoretical model describing the electrostatically driven step of the ␣1b-adrenergic receptor (AR)-G protein recognition. The comparative analysis of the structuraldynamics features of functionally different receptor forms, i.e., the wild type (ground state) and its constitutively active mutants D142A and A293E, was instrumental to gain insight on the receptor-G protein electrostatic and steric complementarity. Rigid body docking simulations between the different forms of the ␣1b-AR and the heterotrimeric G␣q, G␣s, G␣i1, and G␣t suggest that the cytosolic crevice shared by the active receptor and including the second and the third intracellular loops as well as the cytosolic extension of helices 5 and 6, represents the receptor surface with docking complementarity with the G protein. On the other hand, the G protein solvent-exposed portions that recognize the intracellular loops of the activated receptors are the Nterminal portion of ␣3, ␣G, the ␣G/␣4 loop, ␣4, the ␣4/6 loop, ␣5, and the C-terminus. Docking simulations suggest that the two constitutively active mutants D142A and A293E recognize different G proteins with similar selectivity orders, i.e., G␣q Ա G␣s Ͼ G␣i Ͼ Ͼ G␣t. The theoretical models herein proposed might provide useful suggestions for new experiments aiming at exploring the receptor-G protein interface. Proteins 1999;37:145-156.
Proceedings of the National Academy of Sciences, 1990
The adrenergic receptors (ARs) (subtypes alpha 1, alpha 2, beta 1, and beta 2) are a prototypic f... more The adrenergic receptors (ARs) (subtypes alpha 1, alpha 2, beta 1, and beta 2) are a prototypic family of guanine nucleotide binding regulatory protein-coupled receptors that mediate the physiological effects of the hormone epinephrine and the neurotransmitter norepinephrine. We have previously assigned the genes for beta 2- and alpha 2-AR to human chromosomes 5 and 10, respectively. By Southern analysis of somatic cell hybrids and in situ chromosomal hybridization, we have now mapped the alpha 1-AR gene to chromosome 5q32----q34, the same position as beta 2-AR, and the beta 1-AR gene to chromosome 10q24----q26, the region where alpha 2-AR is located. In mouse, both alpha 2- and beta 1-AR genes were assigned to chromosome 19, and the alpha 1-AR locus was localized to chromosome 11. Pulsed field gel electrophoresis has shown that the alpha 1- and beta 2-AR genes in humans are within 300 kilobases (kb) and the distance between the alpha 2- and beta 1-AR genes is less than 225 kb. The proximity of these two pairs of AR genes and the sequence similarity that exists among all the ARs strongly suggest that they are evolutionarily related. Moreover, they likely arose from a common ancestral receptor gene and subsequently diverged through gene duplication and chromosomal duplication to perform their distinctive roles in mediating the physiological effects of catecholamines. The AR genes thus provide a paradigm for understanding the evolution of such structurally conserved yet functionally divergent families of receptor molecules.
Proceedings of the National Academy of Sciences, 1997
To investigate the functional role of different ␣ 1 -adrenergic receptor (␣ 1 -AR) subtypes in vi... more To investigate the functional role of different ␣ 1 -adrenergic receptor (␣ 1 -AR) subtypes in vivo, we have applied a gene targeting approach to create a mouse model lacking the ␣ 1b -AR (␣ 1b ؊͞؊). Reverse transcription-PCR and ligand binding studies were combined to elucidate the expression of the ␣ 1 -AR subtypes in various tissues of ␣ 1b ؉͞؉ and ؊͞؊ mice. Total ␣ 1 -AR sites were decreased by 98% in liver, 74% in heart, and 42% in cerebral cortex of the ␣ 1b ؊͞؊ as compared with ؉͞؉ mice. Because of the large decrease of ␣ 1 -AR in the heart and the loss of the ␣ 1b -AR mRNA in the aorta of the ␣ 1b ؊͞؊ mice, the in vivo blood pressure and in vitro aorta contractile responses to ␣ 1 -agonists were investigated in ␣ 1b ؉͞؉ and ؊͞؊ mice. Our findings provide strong evidence that the ␣ 1b -AR is a mediator of the blood pressure and the aorta contractile responses induced by ␣ 1 agonists. This was demonstrated by the finding that the mean arterial blood pressure response to phenylephrine was decreased by 45% in ␣ 1b ؊͞؊ as compared with ؉͞؉ mice. In addition, phenylephrine-induced contractions of aortic rings also were decreased by 25% in ␣ 1b ؊͞؊ mice. The ␣ 1b -AR knockout mouse model provides a potentially useful tool to elucidate the functional specificity of different ␣ 1 -AR subtypes, to better understand the effects of adrenergic drugs, and to investigate the multiple mechanisms involved in the control of blood pressure.
Proceedings of the National Academy of Sciences, 2007
In response to various pathological stresses, the heart undergoes a pathological remodeling proce... more 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 ␣1-adrenergic receptors (ARs). In particular, our results indicate that suppression of AKAP-Lbc expression by infecting rat neonatal ventricular cardiomyocytes with lentiviruses encoding AKAP-Lbcspecific short hairpin RNAs strongly reduces both ␣1-AR-mediated RhoA activation and hypertrophic responses. Interestingly, ␣1-ARs promote AKAP-Lbc activation via a pathway that requires the ␣ 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.
Proceedings of the National Academy of Sciences, 1997
In this study, a quantitative approach was used to investigate the role of D142, which belongs to... more In this study, a quantitative approach was used to investigate the role of D142, which belongs to the highly conserved E͞DRY sequence, in the activation process of the ␣ 1B -adrenergic receptor (␣ 1B -AR). Experimental and computer-simulated mutagenesis were performed by substituting all possible natural amino acids at the D142 site. The resulting congeneric set of proteins together with the finding that all the receptor mutants show various levels of constitutive (agonist-independent) activity enabled us to quantitatively analyze the relationships between structural͞dynamic features and the extent of constitutive activity. Our results suggest that the hydrophobic͞hydrophilic character of D142, which could be regulated by protonation͞deprotonation of this residue, is an important modulator of the transition between the inactive (R) and active (R*) state of the ␣ 1B -AR. Our study represents an example of quantitative structureactivity relationship analysis of the activation process of a G protein-coupled receptor.
Tumor promoting phorbol esters stimulate Ca/sup + +/ phospholipid-dependent protein kinase C. It ... more Tumor promoting phorbol esters stimulate Ca/sup + +/ phospholipid-dependent protein kinase C. It has been suggested that this enzyme regulates the functional properties of different cell membrane receptors. In this study we investigated the effect of phorbol esters on ..cap alpha..â-adrenoceptor binding and phosphatidylinositol metabolism in cultured smooth muscle cells derived from rabbit aorta. Treatment of these cells with biologically active phorbol esters for 15 min. to 2 hours caused a marked decrease of norepinephrine stimulation of inositol phospholipid metabolism and a 3 fold decrease in agonist affinity for ¹²âµI-HEAT binding to ..cap alpha..â-adrenoceptors in the intact smooth muscle cells. The ability of phorbol esters to modulate ..cap alpha..â-adrenoceptor responsiveness suggests that activation of protein kinase C may represent an important mechanism regulating ..cap alpha..â-adrenergic receptor functional properties. 32 references, 4 figures.
Obesity, 2007
GARRUTI, GABRIELLA, VITTORIO GIUSTI, JÜ RG NUSSBERGER, CHRISTIAN DARIMONT, CHANTAL VERDUMO, CATHE... more GARRUTI, GABRIELLA, VITTORIO GIUSTI, JÜ RG NUSSBERGER, CHRISTIAN DARIMONT, CHANTAL VERDUMO, CATHERINE AMSTUTZ, FRANCESCO PUGLISI, FRANCESCO GIORGINO, RICCARDO GIORGINO, AND SUSANNA COTECCHIA. Expression and secretion of the atrial natriuretic peptide in human adipose tissue and preadipocytes. Obesity. 2007;15: 2181-2189.
Neurobiology of Learning and Memory, 2001
Knockout mice lacking the alpha-1b adrenergic receptor were tested in behavioral experiments. Rea... more Knockout mice lacking the alpha-1b adrenergic receptor were tested in behavioral experiments. Reaction to novelty was first assessed in a simple test in which the time taken by the knockout mice and their littermate controls to enter a second compartment was compared. Then the mice were tested in an open field to which unknown objects were subsequently added. Special novelty was introduced by moving one of the familiar objects to another location in the open field. Spatial behavior and memory were further studied in a homing board test, and in the water maze. The alpha-1b knockout mice showed an enhanced reactivity to new situations. They were faster to enter the new environment, covered longer paths in the open field, and spent more time exploring the new objects. They reacted like controls to modification inducing spatial novelty. In the homing board test, both the knockout mice and the control mice seemed to use a combination of distant visual and proximal olfactory cues, showing place preference only if the two types of cues were redundant. In the water maze the alpha-1b knockout mice were unable to learn the task, which was confirmed in a probe trial without platform. They were perfectly able, however, to escape in a visible platform procedure. These results confirm previous findings showing that the noradrenergic pathway is important for the modulation of behaviors such as reaction to novelty and exploration, and suggest that this is mediated, at least partly, through the alpha-1b adrenergic receptors. The lack of alpha-1b adrenergic receptors in spatial orientation does not seem important in cue-rich tasks but may interfere with orientation in situations providing distant cues only. ᭧
Naunyn-Schmiedeberg's Archives of Pharmacology, 2001
In order to characterize inverse agonism at α 1Badrenoceptors, we have compared the concentration... more In order to characterize inverse agonism at α 1Badrenoceptors, we have compared the concentration-response relationships of several quinazoline and non-quinazoline α 1 -adrenoceptor antagonists at cloned hamster wildtype (WT) α 1B -adrenoceptors and a constitutively active mutant (CAM) thereof upon stable expression in Rat-1 fibroblasts. Receptor activation or inhibition thereof was assessed as [ 3 H]inositol phosphate (IP) accumulation. Quinazoline (alfuzosin, doxazosin, prazosin, terazosin) and non-quinazoline α 1 -adrenoceptor antagonists (BE 2254, SB 216,469, tamsulosin) concentration-dependently inhibited phenylephrine-stimulated IP formation at both WT and CAM with K i values similar to those previously found in radioligand binding studies. At CAM in the absence of phenylephrine, the quinazolines produced concentrationdependent inhibition of basal IP formation; the maximum inhibition was ~55%, and the corresponding EC 50 values were slightly smaller than the K i values. In contrast, BE 2254 produced much less inhibition of basal IP formation, SB 216,469 was close to being a neutral antagonist, and tamsulosin even weakly stimulated IP formation. The inhibitory effects of the quinazolines and BE 2254 as well as the stimulatory effect of tamsulosin were equally blocked by SB 216,469 at CAM. At WT in the absence of phenylephrine, tamsulosin did not cause significant stimulation and none of the other compounds caused significant inhibition of basal IP formation. We conclude that α 1 -adrenoceptor antagonsits with a quinazoline structure exhibit greater efficacy as inverse agonists than those without.
Methods, 1998
Acting either coordinately or independently, these two This work describes the ab initio procedur... more Acting either coordinately or independently, these two This work describes the ab initio procedure employed to build species bind and modulate the activities of downstream an activation model for the a 1b -adrenergic receptor (a 1b -AR). The effector molecules. G proteins are released from effirst version of the model was progressively modified and complifectors on the breakdown of GTP that results from the cated by means of a many-step iterative procedure characterized slow GTPase activity of the a subunit. The inactive a by the employment of experimental validations of the model in subunit can recombine with bg, reforming the heteroeach upgrading step. A combined simulated (molecular dynamtrimer, which can then reassociate with its receptor ics) and experimental mutagenesis approach was used to deterand undergo a new cycle of signal transduction (3). mine the structural and dynamic features characterizing the in-Despite the fact that recent crystallographic studies active and active states of a 1b -AR. The latest version of the of G protein a subunits and heterotrimers have begun Recently, biophysical and biochemical studies on rhodopsin showed that a relative displacement of helices
Life Sciences, 2009
Aim: Alpha1-adrenergic receptors (α 1 -ARs) are classified into three subtypes: α 1A -AR, α 1B -A... more Aim: Alpha1-adrenergic receptors (α 1 -ARs) are classified into three subtypes: α 1A -AR, α 1B -AR, and α 1D -AR. Triple disruption of α 1A -AR, α 1B -AR, and α 1D -AR genes results in hypotension and produces no contractile response of the thoracic aorta to noradrenalin. Presently, we characterized vascular contractility against other vasoconstrictors, such as potassium, prostaglandin F2alpha (PGF 2α ) and 5-hydroxytryptamine (5-HT), in α 1A -AR, α 1B -AR, and α 1D -AR triple knockout (α 1 -AR triple KO) mice. Main methods: The contractile responses to the stimulation with vasoconstrictors were studied using isolated thoracic aorta. Key findings: As a result, the phasic and tonic contraction induced by a high concentration of potassium (20 mM) was enhanced in the isolated thoracic aorta of α 1 -AR triple KO mice compared with that of wild-type (WT) mice. In addition, vascular responses to PGF 2α and 5-HT were also enhanced in the isolated thoracic aorta of α 1 -AR triple KO mice compared with WT mice. Similar to in vitro findings with isolated thoracic aorta, in vivo pressor responses to PGF 2α were enhanced in α 1 -AR triple KO mice. Real-time reverse transcription-polymerase chain reaction analysis and western blot analysis indicate that gene expression of the 5-hydroxytryptamine 2A (5-HT 2A ) receptor was up-regulated in the thoracic aorta of α 1 -AR triple KO mice while the prostaglandin F2α receptor (FP) was unchanged. Significance: These results suggest that loss of α 1 -ARs can lead to enhancement of vascular responsiveness to the vasoconstrictors and may imply that α 1 -ARs and the subsequent signaling regulate the vascular responsiveness to other stimulations such as depolarization, 5-HT and PGF 2α .
Journal of Surgical Research, 1991