Specific, high-affinity binding sites for angiotensin II on Mycoplasma hyorhinis.

Mycoplasmataceae are known to express various proteins that are similar to those present in mammals. We report a strain of Mycoplasma hyorhinis isolated from opossum kidney cells with specific, high-affinity binding sites for human angiotensin II (Kd = 5.1 +/- 1.9 nM). In contrast, two strains of M. hominis revealed no specific binding. These binding sites resembled mammalian angiotensin II receptors by their high affinity and by their sensitivity to dithiothreitol. However, they are different from mammalian angiotensin II receptors in that they bind angiotensin I with high affinity (Kd = 1.6 +/- 0.29 nM) but not angiotensin III (Kd approximately 330,000 nM). [125I]-angiotensin II binding was not inhibited by angiotensin receptor subtype antagonists DuP 753 and CGP 42112A but it was sensitive to bacitracin and aprotinin. Positions Asp1, Ile5, His6 and Pro7 were essential for binding to M. hyorhinis as deletion of these residues led to a more than 10,000-fold decrease in affinity.     

α1-Adrenergic Receptor-Mediated Downregulation of Angiotensin II Receptors in Neuronal Cultures

Previous evidence has suggested that brain catecholamine levels are important in the regulation of central angiotensin II receptors. In the present study, the effects of norepinephrine and 3,4-dihydroxyphenylethylamine (dopamine) on angiotensin II receptor regulation in neuronal cultures from rat hypothalamus and brainstem have been examined. Both catecholamines elicit significant decreases in [125I]angiotensin II-specific binding to neuronal cultures prepared from normotensive rats, effects that are dose dependent and that are maximal within 4-8 h of preincubation. Saturation and Scatchard analyses revealed that the norepinephrine-induced decrease in the binding is due to a decrease in the number of angiotensin II receptors in neuronal cultures, with little effect on the receptor affinity. Norepinephrine has no significant actions on [125I]angiotensin II binding in cultures prepared from spontaneously hypertensive rats. The downregulation of angiotensin II receptors by norepinephrine or dopamine is blocked by alpha 1-adrenergic and not by other adrenergic antagonists, a result suggesting that this effect is initiated at the cell surface involving alpha 1-adrenergic receptors. This is further supported by our data indicating a parallel downregulation of specific alpha 1-adrenergic receptors elicited by norepinephrine. In summary, these results show that norepinephrine and dopamine are able to alter the regulation of neuronal angiotensin II receptors by acting at alpha 1-adrenergic receptors, which is a novel finding.     

3D model for TM region of the AT-1 receptor in complex with angiotensin II independently validated by site-directed mutagenesis data

A three-dimensional model of the complex of angiotensin II (AII) with the transmembrane (TM) region of the angiotensin II receptor of type 1 (the AT-1 receptor) was obtained by molecular modeling procedures employing structural homology to the X-ray structure of rhodopsin. Since the modeling procedure considered only steric and energy considerations without prior knowledge of the experimental results of site-directed mutagenesis, the results with receptor mutants could be used for independent validation of the model. Indeed, the model brings in contact the residues of AII responsible for agonistic activity, Tyr(4), His(6), and Phe(8), with many residues of AT-1 involved in signal transduction according to site-directed mutagenesis. The model also predicts the existence of several possible conformational pathways for transferring the binding signal through the TM region of AT-1 to the intracellular loops interacting with the G-protein.     

Differential affinities of visual arrestin, beta arrestin1, and beta arrestin2 for G protein-coupled receptors delineate two major classes of receptors

Visual arrestin, betaarrestin1, and betaarrestin2 comprise a family of intracellular proteins that desensitize G protein-coupled receptors (GPCRs). In addition, betaarrestin1 and betaarrestin2 target desensitized receptors to clathrin-coated pits for endocytosis. Whether arrestins differ in their ability to interact with GPCRs in cells is not known. In this study, we visualize the interaction of arrestin family members with GPCRs in real time and in live cells using green fluorescent protein-tagged arrestins. In the absence of agonist, visual arrestin and betaarrestin1 were found in both the cytoplasm and nucleus of HEK-293 cells, whereas betaarrestin2 was found only in the cytoplasm. Analysis of agonist-mediated arrestin translocation to multiple GPCRs identified two major classes of receptors. Class A receptors (beta2 adrenergic receptor, mu opioid receptor, endothelin type A receptor, dopamine D1A receptor, and alpha1b adrenergic receptor) bound betaarrestin2 with higher affinity than betaarrestin1 and did not interact with visual arrestin. In contrast, class B receptors (angiotensin II type 1A receptor, neurotensin receptor 1, vasopressin V2 receptor, thyrotropin-releasing hormone receptor, and substance P receptor) bound both betaarrestin isoforms with similar high affinities and also interacted with visual arrestin. Switching the carboxyl-terminal tails of class A and class B receptors completely reversed the affinity of each receptor for the visual and non-visual arrestins. In addition, exchanging the betaarrestin1 and betaarrestin2 carboxyl termini reversed their extent of binding to class A receptors as well as their subcellular distribution. These results reveal for the first time marked differences in the ability of arrestin family members to bind GPCRs at the plasma membrane. Moreover, they show that visual arrestin can interact in cells with GPCRs other than rhodopsin. These findings suggest that GPCR signaling may be differentially regulated depending on the cellular complement of arrestin isoforms and the ability of arrestins to interact with other cellular proteins.     

Characterization of the human beta -glucan receptor and its alternatively spliced isoforms

beta-1,3-d-Glucans are biological response modifiers with potent effects on the immune system. A number of receptors are thought to play a role in mediating these responses, including murine Dectin-1, which we recently identified as a beta-glucan receptor. In this study we describe the characterization of the human homologue of this receptor and show that it is structurally and functionally similar to the mouse receptor. The human beta-glucan receptor is a type II transmembrane receptor with a single extracellular carbohydrate recognition domain and an immunoreceptor tyrosine activation motif in its cytoplasmic tail. The human beta-glucan receptor is widely expressed and functions as a pattern recognition receptor, recognizing a variety of beta-1,3- and/or beta-1,6-linked glucans as well as intact yeast. In contrast to the murine receptor, the human receptor mRNA is alternatively spliced, resulting in two major (A and B) and six minor isoforms. The two major isoforms differ by the presence of a stalk region separating the carbohydrate recognition domain from the transmembrane region and are the only isoforms that are functional for beta-glucan binding. The human receptor also binds T-lymphocytes at a site distinct from the beta-glucan binding site, indicating that this receptor can recognize both endogenous and exogenous ligands.     

Sarcosine1,glycine8 angiotensin II is an AT1 angiotensin II receptor subtype selective antagonist

Studies predating the discovery of the two major subtypes of angiotensin II (Ang II) receptors, AT1 and AT2, revealed anomalous characteristics of sarcosine1,glycine8 Ang II (Sar1,Gly8 Ang II). It competed poorly for 125I-Ang II binding in bovine brain but potently antagonized dipsogenic responses to intracerebroventricularly administered Ang II. Subsequent recognition that bovine brain contains AT(2) receptors, while dipsogenic responses to Ang II are mediated by AT1 receptors, suggests that Sar1,Gly(8) Ang II is AT1 selective. Sar1,Gly8 Ang II competed for 125I-sarcosine1,isoleucine8 Ang II binding to AT1 receptors in pituitary, liver and adrenal (the latter with the AT2 selective antagonist PD 123,319) with Ki's of 0.66, 1.40 and 1.36 nM, respectively. In contrast, the Ki of Sar1,Gly8 Ang II for AT2 receptors in rat adrenal (with the selective AT1 antagonist losartan) was 52 nM. 125I-Sar1,Gly8 Ang II (0.5-3 nM) bound to AT1 receptors in pituitary, liver, heart, adrenal, and hypothalamic membranes with high affinity (Kd=0.43, 1.6, 2.3, 0.96 and 1.8 nM, respectively), but showed no saturable binding to the adrenal AT2 receptor. 125I-Sar1,Gly8 Ang II selectively labeled AT1 receptors in sections of adrenal using receptor autoradiography. Thus, binding studies reveal Sar1,Gly8 Ang II to be the first angiotensin peptide analog to show AT1 receptor selectivity. 125I-Sar1,Gly8 Ang II offers a new means to selectively radiolabel AT1 receptors and may help to characterize ligand docking sites and agonist switches for AT1 versus AT2 receptors.     

Human brain contains a novel non-AT1, non-AT2 binding site for active angiotensin peptides

AIMS: To determine whether the novel non-AT1, non-AT2 binding site for angiotensins recently discovered in rodent brains occurs in the human brain. MAIN METHODS: Radioligand binding assays of (125)I-sarcosine(1), isoleucine(8) angiotensin II binding were carried out in homogenates of the rostral pole of the temporal cortex of human brains containing 0.3 mM parachloromercuribenzoate (PCMB), 10 microM losartan to saturate AT1 receptors, 10 microM PD123319 to saturate AT2 receptors, with or without 10 microM angiotensin II to define specific binding. Competition binding assays employed a variety of angiotensin peptides, specific angiotensin receptor antagonists, several neuropeptides and an endopeptidase inhibitor to determine pharmacological specificity for this binding site. KEY FINDINGS: The novel non-AT1, non-AT2 binding site was present in similar amounts in female and male brains: Bmax 1.77+/-0.16 and 1.52+/-0.17 fmol/mg initial wet weight in female and male brains, respectively. The K(D) values, 1.79+/-0.09 nM for females, and 1.53+/-0.06 nM for males were also similar. The binding site shows pharmacological specificity similar to that in rodent brains: sarcosine(1), isoleucine(8) angiotensin II>angiotensin III>angiotensin II>angiotensin I'angiotensin IV>angiotensin 1-7. Shorter angiotensin fragments and non-angiotensin peptides showed low affinity for this binding site. SIGNIFICANCE: The presence in human brain of this novel non-AT1, non-AT2 binding site supports the concept that this binding site is an important component of the brain angiotensin system. The functional significance of this binding site, either as a novel angiotensin receptor or a highly specific angiotensinase remains to be determined.     

Functionally distinct insulin receptors generated by tissue-specific alternative splicing.

Cloning of the insulin receptor cDNA has earlier revealed the existence of two alternative forms of the receptor differing by the presence or absence of 12 amino acids near the C-terminus of the receptor alpha-subunit. This insert has been shown by others to be encoded by a discrete exon, and alternative splicing of this exon leads to tissue-specific expression of two receptor isoforms. We have studied the functional significance of the receptor isoforms and have confirmed that they are generated by alternative splicing. When cDNAs encoding the two forms of the insulin receptors are expressed in Rat 1 cells, the receptor lacking the insert (HIR-A) has a significantly higher affinity for insulin than the receptor with the insert (HIR-B). This difference in affinity is maintained when insulin binding activity is assayed in solution using detergent solubilized, partially purified receptors. These data, combined with the tissue specificity of HIR-A and HIR-B expression, suggest that alternative splicing may result in the modulation of insulin metabolism or responsiveness by different tissues.     

Cloning and expression of the mouse histamine H3 receptor: evidence for multiple isoforms

The existence of mouse H3-receptor isoforms was investigated by PCR analysis and cDNA cloning. Splicing mechanisms previously reported in various species are conserved in the mouse. The retention/deletion of a fragment in the third intracellular loop of the mouse receptor leads to the existence of three isoforms designated mH(3(445)), mH(3(413)) and mH(3(397)) according to the length of their deduced amino acid sequence. PCR analysis showed that mouse H3-receptor isoforms display different expression patterns in the brain. Following expression in Cos-1 cells, [125I]iodoproxyfan binding indicated similar pharmacological profiles of the mH(3(445)), mH(3(413)) and mH(3(397)) isoforms. The pharmacological profile of the mouse H3 receptor is more similar to the rat receptor than to the human receptor, although some differences were also observed between the mouse and rat receptors. For example, the potency of thioperamide and ciproxifan is slightly higher at the mouse receptor than at the rat receptor but 40-100-fold higher than at the human receptor. In situ hybridization histochemistry showed that the distribution of H3-receptor mRNAs in the mouse brain is rather similar to that previously reported in the rat brain. However, the autoradiographic and cellular expression patterns observed in several brain areas such as the thalamus or hippocampus reveal important differences between the two species.     

Real-time monitoring of the interactions of transforming growth factor-beta (TGF-beta ) isoforms with latency-associated protein and the ectodomains of the TGF-beta type II and III receptors reveals different kinetic models and stoichiometries of binding

Mature transforming growth factor-beta (TGF-beta) is proteolytically derived from the C terminus of a precursor protein. Latency-associated protein (LAP), the N-terminal remnant of the TGF-beta precursor, is able to bind and neutralize TGF-beta. Mature TGF-beta exerts its activity by binding and complexing members of two subfamilies of receptors, the type I and II receptors. In addition to these signaling receptors, TGF-beta can also interact with an accessory receptor termed the type III receptor. Using a surface plasmon resonance-based biosensor (BIAcore), we determined the mechanisms of interaction of four binding proteins (LAP, the type II and III receptor ectodomains (EDs), and a type II receptor ED/Fc chimera) with three TGF-beta isoforms, and we quantified their related kinetic parameters. Using global fitting based on a numerical integration data analysis method, we demonstrated that LAP and the type II receptor/Fc chimera interacted with the TGF-beta isoforms with a 1:1 stoichiometry. In contrast, the type II ED interactions with TGF-beta were best fit by a kinetic model assuming the presence of two independent binding sites on the ligand molecule. We also showed that the type III ED bound two TGF-beta molecules. Further experiments revealed that LAP was able to block the interactions of TGF-beta with the two EDs, but that the two EDs did not compete or cooperate with each other. Together, these results strongly support the existence of a cell-surface complex consisting of one type III receptor, two TGF-beta molecules, and four type II receptors, prior to the recruitment of the type I receptor for signal transduction. Additionally, our results indicate that the apparent dissociation rate constants are more predictive of the neutralizing potency of these TGF-beta-binding proteins (LAP, the type II and III receptor EDs, and the type II receptor/Fc chimera) than the apparent equilibrium constants.     

Dominance of type 1 angiotensin II receptor in the nonpregnant and pregnant bovine uterus

The present study was undertaken to characterize, determine and localize angiotensin II receptors in the nonpregnant and pregnant bovine uterus. In addition, the concentration of active renin, which is responsible for the generation of angiotensin, was determined. Autoradiography and angiotensin II receptor binding studies showed that all compartments of the bovine uterus contained high concentrations of angiotensin II receptors. In general, the type 1 angiotensin II receptor (AT1) predominated over the AT2 receptor. In the endometrium, the highest density was found in the caruncles and the AT1 receptor was always predominant. The density of angiotensin II receptors in the endometrium increased at the beginning of pregnancy, but decreased and reached values similar to those in nonpregnant animals near term. In the myometrium, the density of angiotensin II receptors was highest at or near the endometrial-myometrial junction. In this area, the predominant type of angiotensin II receptor in the uterus of cyclic cows varied, whereas the AT1 receptor always predominated during pregnancy. Non-AT1 and non-AT2 binding sites were found in the same locations as the angiotensin II receptors, but at lower densities. With the exception of the pregnant endometrium, all compartments contained higher active renin concentrations than found in plasma, indicating local synthesis of renin. This study demonstrates a difference in the expression of types of angiotensin II receptor in the bovine uterus compared with other species. The high densities of angiotensin II receptors localized in several important areas imply that the renin-angiotensin system participates in regulation of growth and tissue function in the bovine uterus.     

Characterization of a novel Eph receptor tyrosine kinase, EphA10, expressed in testis

In mammals, 14 members of the Eph receptor tyrosine kinase family have been described so far. Here we present a not yet described member of this family denoted EphA10. We report the identification of three putative EphA10 isoforms: one soluble and two transmembrane isoforms. One of the latter isoforms lacked the sterile alpha motif commonly found in Eph receptors. The gene encoding EphA10 is located on chromosome 1p34 and expression studies show that EphA10 mRNA is mainly expressed in testis. Binding studies to ephrin ligands suggests that this receptor belongs to the EphA subclass of Eph receptors binding mainly to ephrin-A ligands.     

Transgenic mouse models of angiotensin receptor subtype function in the cardiovascular system

Angiotensin II mediates is biological actions via different subtypes of G protein-coupled receptors, termed AT(1) and AT(2) receptors. In rodents, two AT(1) receptors have been identified, AT(1A) and AT(1B), whereas in humans a single AT(1) receptor exists. Recently, a number of transgenic animal models have been generated which overexpress or lack functional angiotensin II receptor subtypes. This review focuses on the physiological significance of angiotensin II receptor subtype diversity in the cardiovascular system. In the mouse, AT(1A) receptors are the major regulators of cardiovascular homeostasis by determining vascular tone and natriuresis. In addition, AT(1A) receptors mediate growth-stimulating signals in vascular and cardiac myocytes. AT(1B) receptors participate in blood pressure regulation, and their functions become apparent when the AT(1A) receptor gene is deleted. Deletion of the mouse gene for the AT(2) receptor subtype led to hypersensitivity to pressor and antinatriuretic effects of angiotensin II in vivo, suggesting that the AT(2) receptor subtype counteracts some of the biological effects of AT(1) receptor signalling.     

ethylene receptor 1 (etr1) Is Sufficient and Has the Predominant Role in Mediating Inhibition of Ethylene Responses by Silver in Arabidopsis thaliana

Ethylene influences many processes in Arabidopsis thaliana through the action of five receptor isoforms. All five isoforms use copper as a cofactor for binding ethylene. Previous research showed that silver can substitute for copper as a cofactor for ethylene binding activity in the ETR1 ethylene receptor yet also inhibit ethylene responses in plants. End-point and rapid kinetic analyses of dark-grown seedling growth revealed that the effects of silver are mostly dependent upon ETR1, and ETR1 alone is sufficient for the effects of silver. Ethylene responses in etr1-6 etr2-3 ein4-4 triple mutants were not blocked by silver. Transformation of these triple mutants with cDNA for each receptor isoform under the promoter control of ETR1 revealed that the cETR1 transgene completely rescued responses to silver while the cETR2 transgene failed to rescue these responses. The other three isoforms partially rescued responses to silver. Ethylene binding assays on the binding domains of the five receptor isoforms expressed in yeast showed that silver supports ethylene binding to ETR1 and ERS1 but not the other isoforms. Thus, silver may have an effect on ethylene signaling outside of the ethylene binding pocket of the receptors. Ethylene binding to ETR1 with silver was ～30% of binding with copper. However, alterations in the K(d) for ethylene binding to ETR1 and the half-time of ethylene dissociation from ETR1 do not underlie this lower binding. Thus, it is likely that the lower ethylene binding activity of ETR1 with silver is due to fewer ethylene binding sites generated with silver versus copper.     

Complexity of FGF receptors: genetic basis for structural diversity and functional specificity.

Since 1989, the receptors for fibroblast growth factors (FGFs) were cloned and characterized as a subgroup of the family of receptor tyrosine kinases. Four FGF receptor genes were identified, all of which encode membrane-bound glycoproteins containing three immunoglobulin (Ig) -like domains at the extracellular region, where only two of these domains are involved in ligand binding. Three unique features characterize the FGF receptors: 1) overlapping recognition and redundant specificity, where one receptor may bind with a similar affinity several of the seven known FGFs and one FGF may bind similarly to several distinct receptors. 2) The binding of FGFs to their receptors is dependent on the interaction of FGF with cell surface heparan sulfate proteoglycans. 3) A multitude of isoforms of cell-bound or secreted receptors are produced by the same gene. The gene structure of these receptors revealed two major mechanisms that are responsible for the formation of the diverse forms: alternative mRNA splicing, resulting in deletions or alternate exons usage, and internal polyadenylation, resulting in truncated products. These are reminiscent of mechanisms that also operate in the immunoglobulin family to generate diversity and to produce either secreted or cell-bound molecules. Tissue-specific alternative splicing in FGF receptors allows for the generation of two distinct receptors from a single gene because alternative exons determine the sequence of the COOH-terminal half of the third Ig-like domain involved in ligand binding. This represents a novel genetic mechanism to generate receptor diversity and specificity and to increase receptor repertoire.     

Differential beta-arrestin binding of AT1 and AT2 angiotensin receptors

Agonist stimulation of G protein-coupled receptors causes receptor activation, phosphorylation, beta-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 beta-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.     

Polymorphism and signalling of melatonin receptors.

Melatonin receptors belong to the superfamily of G protein-coupled receptors. Cloning of Mel1c receptors expressed in Xenopus skin revealed the existence of a polymorphism for these receptors. Heterologous expression of the two allelic isoforms, called Mel1c(alpha) and Mel1c(beta), indicated functional differences in their signalling properties. Both isoforms are coupled to the cAMP and cGMP pathways. However, the alpha isoform is preferentially coupled to the cAMP pathway, whereas the beta isoform couples preferentially to the cGMP pathway. Coupling differences may be explained by the fact that five of the six amino acid substitutions between the two isoforms are localized within intracellular receptor regions potentially involved in G protein coupling. Allelic isoforms were also observed for Mel1a receptors expressed in ovine pars tuberalis, suggesting that polymorphism is a general feature of the melatonin receptor family. We also evaluated the potential of the two human melatonin receptor subtypes, Mel1a and Mel1b, to modulate the cGMP pathway. Melatonin inhibited intracellular cGMP levels in a dose-dependent manner in HEK293 cells transfected with the human Mel1b receptor. This was not the case for HEK293 cells transfected with the human Mel1a receptor. In conclusion, our results indicate that the expression of receptor subtypes and isoforms may permit differential signalling between melatonin receptors.