Targeted disruption of the mouse beta1-adrenergic receptor gene: developmental and cardiovascular effects

At least three distinct beta-adrenergic receptor (beta-AR) subtypes exist in mammals. These receptors modulate a wide variety of processes, from development and behavior, to cardiac function, metabolism, and smooth muscle tone. To understand the roles that individual beta-AR subtypes play in these processes, we have used the technique of gene targeting to create homozygous beta 1-AR null mutants (beta 1-AR -/-) in mice. The majority of beta 1-AR -/- mice die prenatally, and the penetrance of lethality shows strain dependence. Beta l-AR -/- mice that do survive to adulthood appear normal, but lack the chronotropic and inotropic responses seen in wild-type mice when beta-AR agonists such as isoproterenol are administered. Moreover, this lack of responsiveness is accompanied by markedly reduced stimulation of adenylate cyclase in cardiac membranes from beta 1-AR -/- mice. These findings occur despite persistent cardiac beta 2-AR expression, demonstrating the importance of beta 1-ARs for proper mouse development and cardiac function, while highlighting functional differences between beta-AR subtypes.     

Distribution of myocardial beta-adrenoceptor subtypes and coupling to the adenylate cyclase in children with congenital heart disease and implications for treatment

In congestive heart failure, down-regulation of myocardial beta-adrenoceptors (beta-AR) due to an elevated sympathetic tone is well known. In infancy and childhood, heart failure is usually related to congenital heart disease (CHD). Therefore, 71 samples of right atrial tissue of infants and children with CHD undergoing cardiac surgery were studied for beta-adrenoceptor density and distribution of the beta 1-/beta 2-AR subtypes. In 49 cases, the coupling of the beta-AR to the adenylate cyclase (AC) was examined. In a further study of 19 myocardial samples, AC was selectively stimulated with beta 1- or beta 2-AR whereas the other subtype was blocked by an antagonist. The following results were obtained: (1) Infants and children with severe acyanotic or cyanotic CHD had severely reduced beta-AR densities. (2) In most of the cases, the beta-AR down regulation is beta 1-subtype selective, but in critically ill newborns with congenital aortic valve stenosis or transposition of the great arteries, there is additional significant beta 2-AR down-regulation. In Fallot patients treated with the beta-antagonist propranolol, a significant increased beta-AR number compared with untreated Fallot patients was found. (3) beta-Adrenoceptor reduction in CHD is correlated with elevated noradrenaline plasma levels, thus proving a sympathetic dysregulation. (4) In CHD with moderate hemodynamic load, beta 2-AR coupling to AC was markedly more efficient than beta 1-AR coupling. The small number of myocardial beta 2-AR produced most of the cyclic adenosine monophosphate. (5) In severe acyanotic and cyanotic CHD, a partial decoupling of the beta 2-AR to the AC occurred.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of beta-adrenergic receptors of freshly isolated astrocytes and neurons from rat brain

The binding and characteristics of rat brain beta-adrenergic receptors (beta-AR) isolated from astrocytes and neurons were investigated. Equilibrium binding experiments demonstrated that beta-AR were more concentrated on astrocytes than on neurons isolated from forebrain, cerebral cortex and cerebellum. Inhibition experiments revealed that beta 1-AR and beta 2-AR were present in the two cell types. Isoproterenol revealed two interchangeable states of high and low affinity binding to both beta 1- and beta 2-AR in neurons. The high affinity binding sites were sensitive to guanylylimidodiphosphate (GppNHp). Similar results were found with other beta-AR agonists but not with salbutamol and salmeterol which recognized both affinity states of the neuronal beta 2-AR but only the low affinity state of beta 1-AR. In astrocytes only the low affinity state of beta-AR was observed.     

Regulation of endothelial permeability by beta-adrenoceptor agonists: contribution of beta 1- and beta 2-adrenoceptors

The barrier function of cultured, macrovascular endothelial cells derived from bovine aorta was analyzed using confluent monolayers of cells and measuring the exchange of fluorescein dextrans of different molecular masses. The effects of beta-adrenoceptor agonists with different selectivity for beta 1- and beta 2-adrenoceptors (AR) were investigated. Formoterol, a novel high-affinity agonist for beta 2-AR recently introduced in the treatment of bronchial asthma, showed a significant reduction of cell permeability with subnanomolar concentrations, whereas the catecholamines (-)-isoproterenol and (-)-norepinephrine only showed significant effects with micromolar concentrations. In order to elucidate if this difference in potential to regulate cell permeability is related to appropriate changes in the selectivity and affinity of the agonists for beta 2 AR, we investigated the beta AR-coupled adenylate cyclase (AC) in membranes from endothelial cells and compared AC stimulation with the binding of agonists to the receptors using [125I](-)-iodopindolol as radioligand. beta-Adrenoceptors revealed to be closely coupled to AC as assessed by a similar magnitude of effects by receptor agonists in comparison to GTP analogues and direct stimulants of AC activity. AC activity was increased by formoterol in parallel to its receptor occupancy of beta 2AR with nanomolar concentrations which were 50-fold higher than those used for the regulation of cell permeability indicating the existence of spare receptors. In contrast to formoterol, the catecholamines (-)-isoproterenol and (-)-norepinephrine stimulated AC activity through both beta 1AR and beta 2AR. From the overproportional high contribution of beta 1AR to AC stimulation (42%) in comparison to its low fraction (13%) in receptor binding we calculated that beta 1AR is 3-4-fold more effectively coupled to AC than beta 2 AR.     

Beta-3 adrenergic receptor agonists cause an increase in gastrointestinal transit time in wild-type mice, but not in mice lacking the beta-3 adrenergic receptor

The effects of beta-3 adrenergic receptor (beta3-AR) agonists on gastrointestinal (GI) motility, as reported by stomach retention and intestinal transit of radiolabelled charcoal, were compared in wild-type (WT) mice and in transgenic mice lacking beta3-AR (beta3-AR[KO]) or having beta3-AR in white and brown adipose tissue only (beta3-AR[WAT+BAT]). After s.c. administration of 3 mg/kg of the selective, rodent specific beta3-AR agonists BRL 35135, CL 316, 243 or ICI 198,157, WT mice exhibited a significant decrease in the extent of movement of radiotracer through the stomach and intestines, indicative of decreased GI motility. These compounds also caused an increase in plasma glycerol levels in the WT mice, suggesting that increased lipolysis in adipose tissue had been evoked. None of these compounds had an effect on GI motility or evoked lipolysis in the beta3-AR[KO] mice. Treatment of WT mice with SR 56811A, a beta3-AR agonist that exhibited a relatively lower affinity for rodent beta3-AR in vitro, did not affect GI motility or plasma glycerol levels in WT or beta3[KO] mice when administered s.c. at 3 mg/kg. Clonidine, an alpha-2 adrenergic receptor agonist, used as a positive control in these GI studies, caused a decrease in GI motility in both WT and beta3-AR[KO] mice. These results are consistent with a postulated role for beta3-AR in regulation of GI motility in the mouse. However, treatment of beta3-AR[WAT+BAT] mice with 3 mg/kg BRL 35135 resulted in elevated plasma glycerol levels, as well as increased stomach retention and decreased intestinal transit of radiotracer. These results suggest that this beta3-AR agonist may exert its effects on the GI tract indirectly, through an unknown signaling mechanism activated by agonism of beta3-AR in adipose tissue.     

Regulation of the mRNA-binding protein AUF1 by activation of the beta-adrenergic receptor signal transduction pathway

In both cell culture based model systems and in the failing human heart, beta-adrenergic receptors ( beta-AR) undergo agonist-mediated down-regulation. This decrease correlates closely with down-regulation of its mRNA, an effect regulated in part by changes in mRNA stability. Regulation of mRNA stability has been associated with mRNA-binding proteins that recognize A + U-rich elements within the 3'-untranslated regions of many mRNAs encoding proto-oncogene and cytokine mRNAs. We demonstrate here that the mRNA-binding protein, AUF1, is present in both human heart and in hamster DDT1-MF2 smooth muscle cells and that its abundance is regulated by beta-AR agonist stimulation. In human heart, AUF1 mRNA and protein was significantly increased in individuals with myocardial failure, a condition associated with increases in the beta-adrenergic receptor agonist norepinephrine. In the same hearts, there was a significant decrease (approximately 50%) in the abundance of beta1-AR mRNA and protein. In DDT1-MF2 cells, where agonist-mediated destabilization of beta2-AR mRNA was first described, exposure to beta-AR agonist resulted in a significant increase in AUF1 mRNA and protein (approximately 100%). Conversely, agonist exposure significantly decreased (approximately 40%) beta2-adrenergic receptor mRNA abundance. Last, we demonstrate that AUF1 can be immunoprecipitated from polysome-derived proteins following UV cross-linking to the 3'-untranslated region of the human beta1-AR mRNA and that purified, recombinant p37AUF1 protein also binds to beta1-AR 3'-untranslated region mRNA.     

Mice expressing human but not murine beta3-adrenergic receptors under the control of human gene regulatory elements

Beta-adrenergic receptors (ARs) are expressed predominantly in adipose tissue, and beta3-selective agonists are effective anti-obesity drugs in rodents. Rodent and human beta3-ARs differ with respect to expression in white versus brown adipocytes as well as their ability to be stimulated by beta3-AR-selective agonists. Humans express beta3-AR mRNA abundantly in brown but not white adipocytes, while rodents express beta3-AR mRNA abundantly in both sites. To determine the basis for this difference, we have transgenically introduced 74 kilobases (kb) of human beta3-AR genomic sequence into gene knockout mice lacking beta3-ARs. Importantly, human beta3-AR mRNA was expressed only in brown adipose tissue (BAT) of transgenic mice, with little or no expression being detected in white adipose tissue (WAT), liver, stomach, small intestine, skeletal muscle, and heart. This pattern of expression differed from that observed in mice bearing a murine beta3-AR genomic transgene in which beta3-AR mRNA was expressed in both WAT and BAT, but not in other sites. Furthermore, we have transgenically introduced smaller human constructs containing -14.5 and -0.6 kb of upstream sequence into beta3-AR gene knockout mice. Both -14.5 and -0.6 kb constructs were expressed in BAT but not WAT. Thus, human but not murine cis-regulatory elements direct beta3-AR gene expression preferentially to brown adipocytes. Identification of responsible cis-regulatory element(s) and relevant trans-acting factor(s) should provide insight into mechanisms controlling human beta3-AR gene expression. In addition, the beta3-AR agonist, CGP-12177, stimulated oxygen consumption in mice expressing human but not murine beta3-ARs by 91% compared with only 49% in control beta3-AR gene knockout mice, demonstrating that the human beta3-AR can functionally couple with energy expenditure. These "humanized" mice should assist us in the development of drugs that may become effective anti-obesity agents in humans.     

Physiological consequences of beta-adrenergic receptor disruption

Activation of beta-adrenergic receptors (beta-ARs) in vivo is an important means by which animals regulate cardiac performance, vascular tone, lipid and carbohydrate metabolism, and behavior. The advent of targeted gene disruption in mice has led to significant advances in our understanding of the role that beta-AR subtypes play in these processes, and this technique has become an important tool for the study of G protein coupled receptors in general. To date, targeted disruption of both beta1- and beta3-ARs in mice has been reported. Mice lacking beta1-ARs are unresponsive to cardiac beta-AR stimulation, suggesting that neither beta2- nor beta3-ARs couple to inotropic or chronotropic responses in the mouse. Conversely, mice lacking beta3-ARs retain at least some adipose beta-AR responsiveness through remaining beta1- and beta2-ARs, suggesting that all three beta-AR subtypes mediate similar functions in this tissue. While these knockout models have been extremely valuable tools for revealing the roles that individual beta-ARs play in whole animal physiology, it is also useful to integrate the results of experiments derived from either transgenic overexpression of beta-ARs or purely pharmacological approaches to the study of beta-AR function in order to create a comprehensive model of beta-AR function in vivo.     

Transition probability cell cycle model. Part I--Balanced growth

In this study, we have identified and characterized functional alpha2-adrenergic receptor (alpha2-AR) subtypes in human corpus cavernosum and in cultured human corpus cavernosum smooth muscle cells. Analysis of total RNA, isolated from whole corpus cavernosum tissue and smooth muscle cells, by RNase protection assays, demonstrated expression of mRNA for alpha2A, alpha2B, and alpha2C adrenergic receptor subtypes in whole tissue and alpha2A and alpha2C subtypes in cultured smooth muscle cells. Binding studies with [3H]RX821002 (a highly selective and specific ligand for alpha2-adrenergic receptor) in isolated membrane fractions of human corpus cavernosum smooth muscle cells, demonstrated specific alpha2-AR binding sites with high affinity (Kd = 0.63 nM) and limited capacity (25-30 fmol/mg protein). Binding of [3H]RX821002 was displaced with the nonselective alpha-AR antagonist, phentolamine, and with the alpha-AR agonist, norepinephrine, in a dose-dependent manner, but not by the selective alpha1-AR agonist, phenylephrine. Binding of [3H]rauwolscine was also displaced by phentolamine. UK 14,304, a selective alpha2-AR agonist, inhibited forskolin-induced cyclic adenosine monophosphate (cAMP) synthesis in cultured human corpus cavernosum smooth muscle cells and induced dose-dependent contractions of tissue strips in organ bath chambers. UK 14,304-induced contractions were inhibited with alpha2-AR selective antagonists, rauwolscine and delquamine (RS 15385-197). These observations suggest that in human corpus cavernosum, norepinephrine (NE) and epinephrine may activate postsynaptic alpha2-AR subtypes, in addition to activating alpha1-AR subtypes, on smooth muscle cells, contributing to local control of human corpus cavernosum smooth muscle tone, in vivo.     

The contribution of classical (beta1/2-) and atypical beta-adrenoceptors to the stimulation of human white adipocyte lipolysis and right atrial appendage contraction by novel beta3-adrenoceptor agonists of differing selectivities

The role of beta3- and other putative atypical beta-adrenoceptors in human white adipocytes and right atrial appendage has been investigated using CGP 12177 and novel phenylethanolamine and aryloxypropanolamine beta3-adrenoceptor (beta3AR) agonists with varying intrinsic activities and selectivities for human cloned betaAR subtypes. The ability to demonstrate beta1/2AR antagonist-insensitive (beta3 or other atypical betaAR-mediated) responses to CGP 12177 was critically dependent on the albumin batch used to prepare and incubate the adipocytes. Four aryloxypropanolamine selective beta3AR agonists (SB-226552, SB-229432, SB-236923, SB-246982) consistently elicited beta1/2AR antagonist-insensitive lipolysis. However, a phenylethanolamine (SB-220646) that was a selective full beta3AR agonist elicited full lipolytic and inotropic responses that were sensitive to beta1/2AR antagonism, despite it having very low efficacies at cloned beta1- and beta2ARs. A component of the response to another phenylethanolamine selective beta3AR agonist (SB-215691) was insensitive to beta1/2AR antagonism in some experiments. Because no [corrected] novel aryloxypropanolamine had a beta1/2AR antagonist-insensitive inotropic effect, these results establish more firmly that beta3ARs mediate lipolysis in human white adipocytes, and suggest that putative 'beta4ARs' mediate inotropic responses to CGP 12177. The results also illustrate the difficulty of predicting from studies on cloned betaARs which betaARs will mediate responses to agonists in tissues that have a high number of beta1- and beta2ARs or a low number of beta3ARs.     

Prostaglandin E receptor subtypes in cultured rat microglia and their role in reducing lipopolysaccharide-induced interleukin-1beta production

Prostaglandins (PGs) are potent modulators of brain function under normal and pathological conditions. The diverse effects of PGs are due to the various actions of specific receptor subtypes for these prostanoids. Recent work has shown that PGE2, while generally considered a proinflammatory molecule, reduces microglial activation and thus has an antiinflammatory effect on these cells. To gain further insight to the mechanisms by which PGE2 influences the activation of microglia, we investigated PGE receptor subtype, i.e., EP1, EP2, EP3, and EP4, expression and function in cultured rat microglia. RT-PCR showed the presence of the EP1 and EP2 but not EP3 and EP4 receptor subtypes. Sequencing confirmed their identity with previously published receptor subtypes. PGE2 and the EP1 agonist 17-phenyl trinor PGE2 but not the EP3 agonist sulprostone elicited reversible intracellular [Ca2+] increases in microglia as measured by fura-2. PGE2 and the EP2/EP4-specific agonists 11-deoxy-PGE1 and 19-hydroxy-PGE2 but not the EP4-selective agonist 1-hydroxy-PGE1 induced dose-dependent production of cyclic AMP (cAMP). Interleukin (IL)-1beta production, a marker of activated microglia, was also measured following lipopolysaccharide exposure in the presence or absence of the receptor subtype agonists. PGE2 and the EP2 agonists reduced IL-1beta production. IL-1beta production was unchanged by EP1, EP3, and EP4 agonists. The adenylyl cyclase activator forskolin and the cAMP analogue dibutyryl cAMP also reduced IL-1beta production. Thus, the inhibitory effects of PGE2 on microglia are mediated by the EP2 receptor subtype, and the signaling mechanism of this effect is likely via cAMP. These results show that the effects of PGE2 on microglia are receptor subtype-specific. Furthermore, they suggest that specific and selective manipulation of the effects of PGs on microglia and, as a result, brain function may be possible.     

Comparison between beta 3 and beta 2 adrenoceptor agonists as inhibitors of gastric acid secretion

In order to investigate the role of beta 3 adrenoceptors in the regulation of gastric acid secretion we studied the effects of compound SR58611A (a selective agonist for atypical beta adrenoceptors), alone or in combination with beta-adrenoceptor antagonists, in the gastric fistula of a conscious cat. The effects of SR58611A were compared with those of clenbuterol, a selective agonist for beta 2 adrenoceptors. Intravenous infusion of SR58611A (0.3-3 mumol/kg/h) caused a dose-dependent, but partial, inhibition of the acid secretory response to 2-deoxy-D-glucose 100 mg/kg i.v., maximum effect not exceeding 40%. Clenbuterol (0.03-0.1 mumol/kg/h) caused a similar effect (maximum inhibition about 50%) at doses approximately 30 times lower. The acid secretion induced by the histamine H2-receptor agonist dimaprit (1 mumol/kg/h) was minimally affected by both beta adrenoceptor agonists. The inhibitory effect of SR58611A (3 mumol/kg/h) on 2-deoxy-D-glucose-induced acid secretion was not modified by pretreatment with the non-selective beta 1- and beta 2-adrenoceptor blocker propranolol, administered at doses (1.5 mumol/kg iv) that completely blocked the inhibitory effect of clenbuterol (0.1 mumol/kg/h). In contrast, bupranolol (10 mumol/kg i.v.) (a drug endowed with beta 3 antagonistic properties) prevented the inhibitory effects of both SR58611A and clenbuterol. The present data provide functional evidence that, besides beta 2-, also beta 3-adrenoceptors can have negative effects on gastric acid secretion, particularly when it is stimulated by indirect stimuli, like 2-deoxy-D-glucose. This gastric antisecretory activity may represent an additional mechanism for the physio-pharmacological control of gastric acid secretion.     

Binding of a catechol derivative of denopamine (T-0509) and N-tert-butylnoradrenaline (Colterol) to beta 1- and beta 2-adrenoceptors

The affinities for beta-adrenoceptors, the subtype-selectivity and the agonistic effectiveness of T-0509 (a catechol derivative of denopamine) and colterol (N-tert-butylnoradrenaline; Col) were compared with those of other beta-agonists using a binding assay method. Specific binding of [3H]dihydroalprenolol (3H-DHA) to guinea pig left ventricular and lung membranes was saturable, and Scatchard and Hill analyses suggested that 3H-DHA bound to both membranes with a single population of binding sites with no binding site cooperativity. Addition of 5'-guanylylimidodiphosphate (GppNHp, 30 microM) led to a rightward shift of the 3H-DHA binding displacement curves of T-0509 and Col in both membranes, and the degree of shift was similar to that of full agonists such as isoproterenol (Iso), adrenaline (Adr) and noradrenaline (NA). Both T-0509 and Col were thus considered to be full agonists at both beta 1- and beta 2-adrenoceptors, respectively, unlike denopamine and procaterol. T-0509 and Col showed considerably high affinity for both beta 1- and beta 2-adrenoceptors, and T-0509, like denopamine, was as selective for the beta 1-subtype as NA (4.5-fold compared with Iso as a non-selective agonist), whereas Col was more selective for the beta 2-subtype than Adr (4.5-fold compared with Iso).     

Action potential shortening through the putative beta4-adrenoceptor in ferret ventricle: comparison with beta1- and beta2-adrenoceptor-mediated effects

The electrophysiological responses to (-)-CGP 12177 ((-)-4-(3-tertiarybutylamino-2-hydroxypropoxy) benzimidazol-2-one), an agonist for the putative beta4-adrenoceptor, were investigated on isolated perfused ferret hearts paced at 100 min(-1) and compared to those of (-)-noradrenaline and (-)-adrenaline, mediated through beta1- and beta2-adrenoceptors respectively. The three agonists decreased ventricular monophasic action potential duration but prolonged the action potential plateau; beta3-adrenoceptor-selective agonists had no effect. (-)-CGP 12177 was the most potent, but (-)-noradrenaline the most efficacious; both agonists caused ventricular extra-systoles. Because only (-)-noradrenaline but not (-)-CGP 12177 elicited shortening of the refractory period, the mechanism of arrhythmias mediated through beta1- and putative beta4-adrenoceptors may be different.     

Association of beta-agonists with corresponding beta 2- and beta 1-adrenergic pentapeptide sequences

Synthesized beta 1- and beta 2-pentapeptide sequences corresponding to published adrenoceptor transmembrane activation site subtypes were investigated in vitro for selectivity in association for drug ligands of known selectivity. Both nuclear magnetic resonance spectroscopy and molecular mechanics demonstrated that structural differences among the corresponding pentapeptide activation-site sequences can explain agonist selectivity. Results suggest the agonists bind across the activation site loop on the second transmembrane alpha-helix by dipole/dipole interactions between a ligand and the peptide. Since electrostatic interactions within the membrane may determine the rate of intercellular ion flux, agonist association across the activation site sequence could thereby decrease electrostatic resistance to positive ion flux into the cell. Interactions between the peptides and the ligands may provide insight into the structures and mechanisms involved in association of ligands for the identical sequences on the beta-adrenoreceptors.     

The unique nature of the serine interactions for alpha 1-adrenergic receptor agonist binding and activation

Activation of the beta2- and alpha2-adrenergic receptors (AR) involves hydrogen bonding of serine residues in the fifth transmembrane segment (TMV) to the catechol hydroxyls of the endogenous agonists, epinephrine and norepinephrine. With the beta2-AR both Ser204 and Ser207 but not a third TMV serine (Ser203) are required for binding and full agonist activity. However, with the alpha2a-AR only one of two TMV serines (Ser204, equivalent to Ser207 in the beta-AR) appears to contribute partially to agonist-binding and activation. Because the alpha1a-AR uniquely contains only two TMV serines, this subtype was used to systematically evaluate the role of hydrogen bonding in alpha1-AR activation. Binding of epinephrine or its monohydroxyl congeners, phenylephrine and synephrine, was not decreased when tested with alanine- substitution mutants that lacked either Ser188 (Ser188--> Ala) or Ser192 (Ser192-->Ala). With the substitution of both serines in the double mutant, Ser188/192-->Ala, binding of all three ligands was significantly reduced (10- 100-fold) consistent with a single hydrogen bond interaction. However, receptor-mediated inositol phosphate production was markedly attenuated only with the Ser188-->Ala mutation and not with Ser192-->Ala. In support of the importance of Ser188, binding of phenylephrine (meta-hydroxyl only) by Ser192-->Ala increased 7-fold over that observed with either the wild type receptor or the Ser188-->Ala mutation. Binding of synephrine (para-hydroxyl only) was unchanged with the Ser192-->Ala mutation. In addition, when combined with a recently described constitutively active alpha1a-AR mutation (Met292-->Leu), only the Ser188-->Ala mutation and not Ser192-->Ala relieved the high affinity binding and increased agonist potency observed with the Met292-->Leu mutation. A simple interpretation of these findings is that the meta-hydroxyl of the endogenous agonists preferentially binds to Ser188, and it is this hydrogen bond interaction, and not that between the para-hydroxyl and Ser192, that allows receptor activation. Furthermore, since Ser188 and Ser192 are separated by three residues on the TMV alpha-helix, whereas Ser204 and Ser207 of the beta2-AR are separated by only two residues, the orientation of the catechol ring in the alpha1-AR binding pocket appears to be unique and rotated approximately 120 degrees to that in the beta2-AR.     

Discovery of L-755,507: a subnanomolar human beta 3 adrenergic receptor agonist

A study of 4-acylaminobenzenesulfonamides in a cloned human beta 3 adrenergic receptor assay resulted in the discovery of n-hexylurea, L-755,507 (22). This 0.43 nM beta 3 agonist, which is > 440-fold selective over both beta 1 and beta 2 binding, is among the most potent human beta 3 agonists reported to date.     

Antagonist properties of a phosphono isoxazole amino acid at glutamate R1-4 (R,S)-2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid receptor subtypes

All three subtypes of beta-adrenoceptors are coupled to stimulation of adenylyl cyclase activity via the stimulatory guanine-nucleotide-binding protein. Nevertheless, the beta3 adrenoceptor (beta3-AR) differs significantly from the other subtypes in terms of pharmacology. Most strikingly, it recognizes as agonists several compounds acting as potent beta1-AR and beta2-AR antagonists. Furthermore, the human beta3-AR is quite different from the animal beta3-AR. Molecular modelling studies followed by site-directed mutagenesis was used here to identify some of the amino acid residues which may be implicated in ligand binding and signal transduction of the beta3-AR. Three contiguous residues, valine-leucine-alanine, which are present in the first transmembrane domain at positions 48-50 of the human receptor but are absent in all known rodent sequences, were thought to be important for species specificity. When these three residues were deleted from the human receptor, no 'rodent-like' pharmacological profile was obtained in terms of either binding or adenylyl cyclase activation. Glycine at position 53, also in the first transmembrane domain in the human beta3-AR, has been suggested to participate in beta2-/beta3-AR subtype selectivity. Replacement of this glycine residue by phenylalanine, which is the residue present at the homologous position in the human beta2-AR, left the beta3-AR pharmacological profile unaltered in terms of specificity and selectivity. Aspartate residue 117, in the third transmembrane domain, has been found to be essential for ligand binding and consequently adenylyl cyclase activation in several bioamine receptors. When this residue was replaced by a leucine residue in the beta3-AR, ligand binding and signal transduction were suppressed. Finally, replacement of asparagine at position 312 in the sixth transmembrane domain by an alanine residue, led to alterations in the signal-transduction pathway.