Biomedical subjects

M Freissmuth

Publications and source records attributed to M Freissmuth.

At least 55 records · Page 3Linked to original sources

PubMed1995

Species differences in A1 adenosine receptor/G protein coupling: identification of a membrane protein that stabilizes the association of the receptor/G protein complex.

Reconstitution experiments with purified components reproduce the basic characteristics of receptor/G protein coupling, i.e., GTP-sensitive high affinity agonist binding and receptor-promoted GTP binding. However, the interaction of agonists with the A1 adenosine receptor in rat and bovine but not human brain membranes deviates from the ternary complex model since the agonist/receptor/G protein complex cannot be dissociated by high concentrations (> or = 100 microM) of the hydrolysis-resistant analogue GTP gamma S. The reason for this phenomenon referred to as a "tight coupling mode" has remained enigmatic. We show that it is attributable to a distinct membrane protein, which we labeled the coupling cofactor. Extraction of the protein from rat brain membranes with the detergent 3[3-(cholamidopropyl)diamethylammonio]-1-propanamium increased the potency of GTP gamma S by 1000-fold. After extraction, the potency was comparable to that in human brain membrane. Detergent extracts from rat brain membranes were used to resolve the component from solubilized receptors and G protein alpha and beta gamma subunits by sequential DEAE-Sephacel chromatography and Superose gel filtration (molecular weight of approximately 150 kDa in 3[3-(cholamidopropyl)diamethylammonio]-1-propanamium). Coupling cofactor restored guanine nucleotide refractoriness in a concentration-dependent manner to both detergent-extracted rat brain membranes and, albeit with lower affinity, human brain membranes. However, in human brain extracts, cofactor activity was detectable on reconstitution with rat acceptor membranes, indicating an intrinsic difference between rat and human receptors in their ability to interact with the cofactor. With high amounts of coupling cofactor present, GTP gamma S no longer decreased but rather increased agonist affinity. Readdition of partially purified coupling cofactor to acceptor membranes reduced the rate of A1 adenosine receptor-mediated G protein turnover. These observations show that the component identified traps the ternary agonist/receptor/G protein complex in a stable conformation, impedes signaling of the A1 adenosine receptor, and thereby regulates the level of signal amplification.

Animals↗

PubMed1994

Species difference in the G protein selectivity of the human and bovine A1-adenosine receptor.

The purified bovine brain A1-adenosine receptor has previously been shown to discriminate among closely related G protein alpha-subunits. To obtain analogous information for the human receptor, the cDNA coding for the human A1-adenosine receptor was inserted into a plasmid placing the synthesis of the receptor protein under the control of the MalE promoter. Following induction by maltose, active receptor accumulated in Escherichia coli membranes. Binding of the antagonist 8-[3H]cyclopentyl-1,3-dipropylxanthine to E. coli membranes (KD approximately 2 nM, Bmax approximately 0.2-0.4 pmol/mg) showed the appropriate pharmacological profile. Incubation of E. coli membranes with purified Go,i-reconstituted guanine nucleotide-sensitive high affinity binding of the agonist (-)[125I] N6-3-(iodo-4-hydroxyphenylisopropyl)adenosine to the receptor (KD approximately 1 nM). In the presence of purified beta gamma-subunit, the recombinant receptor interacted equally well with the recombinant G protein alpha-subunits Gi alpha-1, Gi alpha-2, Gi alpha-3; G(o) alpha displayed a lower affinity for the receptor while Gs alpha was inactive. Parallel experiments were carried out in bovine and human brain membranes pretreated with N-ethylmaleimide to inactivate the endogenous G(o)/Gi proteins; Gi alpha-3 was most potent in reconstituting 125I-HPIA binding to bovine membranes, while Gi alpha-1, Gi alpha-2, and G(o) alpha displayed similar affinities. However, in human membranes, Gi alpha-1, Gi alpha-2, and Gi alpha-3, were equipotent and high concentrations of G(o) alpha were required to promote 125I-HPIA binding. These observations show (i) that functional human A1-adenosine receptors were synthesized in E. coli; (ii) that the pattern of G protein coupling is identical for the recombinant human A1-receptor and its counterpart in the native membrane; (iii) and that species differences between bovine and human receptor exist not only in their pharmacological profile but also in their G protein specificity suggesting that species homologues of receptors may use different signaling mechanisms.

Animals↗

PubMed1994

2',3'-Dialdehyde GTP as an irreversible G protein antagonist. Disruption and reconstitution of G protein-mediated signal transduction in cells and cell membranes.

The 2',3'-dialdehyde analogue of GTP, oGTP, was devised as an irreversible antagonist of regulatory GTP-binding proteins (G proteins). Here, we show that oGTP uncouples transmembrane signaling mediated by a set of distinct G proteins both in isolated membranes and in whole cells. In human platelet membranes, pretreatment with oGTP suppressed receptor- and G protein-controlled regulation of adenylyl cyclase activity. In chick neuronal cells, inhibition of the voltage-sensitive Ca(2+)-current by various membrane receptors (alpha 2-adrenergic, somatostatin, GABAB) was eliminated when oGTP was applied intracellularly in the whole cell patch-clamp configuration. Disruption of endogenous signaling pathways by oGTP occurred through specific blockage of the GTP-binding site of G protein alpha-subunits by the following criteria: (i) pretreatment of membranes with oGTP blocked direct G protein activation by guanine nucleotides as well as labeling of Gs alpha and Gi alpha with the photoaffinity probe [alpha-32P]GTP azidoanilide. (ii) The effect of oGTP was antagonized by the simultaneous introduction of guanosine 5'-(3-O-thio)triphosphate into the patch-clamped cell. (iii) The time to onset of action was similar for oGTP and guanosine 5'-O-thio)diphosphate. (iv) Inactivation of G protein-dependent signaling was overcome by substituting G protein alpha-subunits. Addition of both the short and long form of recombinant Gs alpha (rGs alpha-s and rGs alpha-L) restored guanine nucleotide-dependent adenylyl cyclase activity to oGTP-treated platelet membranes with rGs alpha-L being approximately 3-10-fold more potent than rGs alpha-s. This apparent preference was due to the intrinsically different activation rates of rGs alpha-L and rGs alpha-s. When reconstituted with exogenous rGs alpha, the A2-adenosine receptor did not discriminate among the two forms of rGs alpha. Thus, Gs alpha-L is the primary determinant of basal cAMP formation in platelets. In contrast, neither the addition of various recombinant subtypes of Gi/o nor purified bovine brain beta gamma-dimers reconstituted adenylyl cyclase inhibition in oGTP-treated membranes. All subtypes of Gi alpha stimulated adenylyl cyclase. In the presence of rGs alpha, a conditional stimulation by beta gamma-dimers was observed. This pattern of stimulation shows that platelet adenylyl cyclase is a type II-like isoform. Either a differently modified G protein or an ancillary GTP-binding component is required for adenylyl cyclase inhibition in platelets. oGTP can be considered a useful tool for disruption and reconstitution of transmembrane signaling mediated by presumably all classes of heterotrimeric G proteins.

Adenylyl Cyclases↗

PubMed1994

Structural and functional characterization of the interaction between 2',3'-dialdehyde guanine nucleotide analogues and the stimulatory G protein alpha-subunit.

We have searched for irreversible ligands which target the guanine nucleotide binding pocket of G protein alpha-subunits by testing the ability of periodate-oxidized 2',3'-dialdehyde guanine nucleotide analogues of GTP (oGTP) and GTP gamma S (oGTP gamma S) to bind to the recombinant alpha-subunit of the stimulatory G protein, rGs alpha-s. oGTP and oGTP gamma S bind to rGs alpha-s in a quasi-irreversible manner via formation of a Schiff's base, which can be reduced with borhydrid resulting in covalent incorporation of [alpha-32P]oGTP and [35S]oGTP gamma S into rGs alpha-s. When bound to rGs alpha-s, oGTP is hydrolyzed and traps the protein in the inactive conformation, while oGTP gamma S persistently activates rGs alpha. Thus, oGTP and oGTP gamma S act as irreversible G protein antagonist and agonist, respectively, and represent a pair of nucleotide analogues suitable as functional and structural tools. Cleavage of covalently labeled rGs alpha-s with cyanogen bromide generates several labeled fragments. Labeled fragments were assigned to the G1 and G4 region of the guanine nucleotide binding pocket using sequence-specific antisera. An additional, labeled fragment was identified by amino-terminal sequencing and corresponded to the helix alpha A in the recently determined crystal structure of the transducin alpha-subunit (Noel, J. P., Hamm, H. E., and Sigler, P. B. (1993) Nature 366, 654-663). In the oGDP-liganded conformation, incorporation occurs predominantly into the G1-fragment, while [35S]oGTP gamma S labels the additional fragments to a similar extent indicating tight packing around the guanine nucleotide binding pocket in the active conformation. Furthermore, rGs alpha-s contains a single acid cleavable bond (Asp317-Pro318), such that formic acid releases a carboxyl-terminal fragment from [alpha-32P]oGTP- and [35S]oGTP gamma S-liganded rGs alpha-s. This fragment contains a single lysine residue (Lys324) which is only labeled by [35S]oGTP gamma S. Lys324 is unique to Gs alpha and lies within its effector binding region. Hence, during the switch from the inactive to the active state, this region undergoes a major conformational change that moves it closer to the nucleotide binding pocket.

Adenylyl Cyclases↗

PubMed1994

Cellular signaling by an agonist-activated receptor/Gs alpha fusion protein.

The consequences of agonist-dependent activation of guanine nucleotide-binding protein (G protein)-coupled receptors vary from cell to cell, depending on a complex network of regulations between components of the signaling cascade. Specific interactions between receptors, G proteins, and effectors are difficult to analyze in intact cells. Engineering of receptor-transducer fusion proteins might be an effective strategy to target cellular effectors more efficiently and specifically. As a model, we evaluated the ability of a fusion protein of beta 2-adrenergic receptor bound to the alpha subunit of adenylyl cyclase-stimulatory G protein (Gs alpha) to restore the defective activation of adenylyl cyclase in S49 cyc- cells that lack endogenous Gs alpha. The coupling between the two partners of the fusion protein was functional, and the agonist-dependent activation of the effector was more potent and more productive in transfected than in wild-type S49 cells. The covalent link between receptor and Gs alpha could thus convey an advantage over freely interacting components. Such receptor-G alpha fusion proteins may help to elucidate the complex interactions between members of signaling pathways and may also constitute a useful tool for studying the effects of single effector activation.

Adenylyl Cyclases↗

PubMed1994

[Antioxidants].

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Antioxidants↗

PubMed1994

Effects of type I-interferons on human thyroid epithelial cells derived from normal and tumour tissue.

Long term interferon (IFN) therapy is frequently associated with side effects which affect the thyroid gland such as hypothyroidism and thyroiditis. We have therefore tested the ability of type I-IFNs to exert direct effects on primary cultures of human thyroid epithelial cells: (i) Type I-IFNs (IFN-alpha 2b and IFN-omega) inhibit cell proliferation as determined by [3H]thymidine incorporation with a half-maximal effect at approximately 1 ng/ml (50 pM). Inhibition of cell growth is observed in cells derived from normal thyroid as well as neoplastic tissue (autonomous and non-secreting adenoma; follicular, papillary and anaplastic carcinoma). (ii) Over a similar concentration range, type I-IFNs suppressed thyroglobulin release by thyroid cells. (iii) IFN-alpha 2b stimulated surface expression of major histocompatibility class (MHC) I but not MHC II molecules, while IFN-gamma enhanced the expression of both MHC I and MHC II molecules. This effect of IFN-gamma, but not that of IFN-alpha 2b was antagonized by suramin. (iv) Incubation of thyroid cells with IFN-alpha 2b also resulted in increased cell surface levels of the intercellular adhesion molecule 1 (ICAM-1). These findings demonstrate that type I-IFNs directly affect thyroid function and explain related side effects of these cytokines. In addition, our results provide a rational basis for the possible use of type I-IFNs in the treatment of patients with advanced thyroid cancer for whom no therapeutic alternative exists.

Cell Division↗

PubMed1994

Reconstructive surgery after hemilaryngectomy for recurrent thyroid cancer using a free jejunal patch-graft.

This case report describes the third recurrence of a poorly differentiated follicular thyroid carcinoma that led to destruction of the thyroid cartilage. Curative surgery required hemilaryngectomy. Because of three previous operations (including neck dissection) and repeated external radiation, the typical laryngoplastic procedures and muscle flaps were not applicable. We therefore employed a stretched jejunal patch to cover the defect so that the neoglottic slit rendered respiration and phonation possible. Thus this approach resulted in functional reconstruction and obviated the need for a tracheostoma. We conclude that the use of a jejunal transplant represents a viable alternative to local reconstruction, particularly in cases complicated by previous surgery and external radiation.

Adenocarcinoma, Follicular↗

PubMed1994

Growth hormone-prolactin-thyrotropin-secreting pituitary adenoma in atypical McCune-Albright syndrome with functionally normal Gs alpha protein.

The McCune-Albright syndrome (MAS) comprises a triad of physical signs: localized bone lesions termed polyostotic fibrous dysplasia, café-au-lait pigmentation of the skin, and autonomous hyperfunction of multiple endocrine systems, including overproduction of GH and T4. A somatic activating point mutation in the gene for the alpha-subunit of the G-protein (Gs alpha) in the affected tissue has been claimed to be the underlying defect. A 29-yr-old patient with MAS, showing polyostotic fibrous dysplasia associated with acromegalic features, underwent endocrinological studies, including oral glucose tolerance test and pituitary stimulation test, and magnetic resonance imaging, revealing elevated plasma concentrations of GH, PRL, and secondary hyperthyroidism due to pituitary macroadenoma infiltrating the sphenoid cavity and extending to the suprasellar space. Subsequently, reduction of tumor mass by a transsphenoidal and a subsequent subfrontal operation led to only marginal amelioration of the excessive hormone production. Postsurgery octreotide and bromocriptine therapy induced near-normalization of hormone concentrations. Immunohistochemistry of tumor tissue confirmed the plurihormonal character, but DNA sequence analysis did not detect any of the two known activating mutations in the Gs alpha gene. Furthermore, biochemical tests revealed normal Gs alpha function, ruling out other mutations that lead to constitutive Gs alpha activation. Our study documents that MAS is a heterogeneous disease. Some, but clearly not all, patients have oncogenic mutations of the gene coding for Gs alpha. Any gene acting down-stream of Gs can theoretically be predicted to result in the same phenotype. In addition, hyperthyroidism of MAS may be secondary to a TSH-producing pituitary macroadenoma.

Adenoma↗

PubMed1994

Thyroid surgery in untreated severe hyperthyroidism: perioperative kinetics of free thyroid hormones in the glandular venous effluent and peripheral blood.

BACKGROUND AND METHODS: It is generally believed that thyroid surgery in Graves' disease requires a euthyroid state to avoid thyrotoxic reactions. We carried out a prospective study on 23 patients who had severe hyperthyroidism with free thyroid hormone concentrations (fT3 or fT4) exceeding the upper normal boundary by 300% or more and who were not pretreated with thyrostatic agents. We determined hormone levels during operation in the thyroid venous effluent before and after surgical trauma and monitored their postoperative elimination kinetics. RESULTS: The concentration of fT3 and fT4 in the venous effluent of the hyperactive gland did not exceed the peripheral levels. Surgery did not induce any intraoperative or postoperative increase in fT4 or fT3, whereas thyroglobulin concentrations rose sharply. Both fT4 and fT3 followed biphasic elimination kinetics, and a significant decline of circulating free hormone concentrations was measurable within the first postoperative hour. CONCLUSIONS: Contrary to widely held assumptions, the surgical trauma does not stimulate the release of thyroid hormones. Hence this mechanism cannot account for the postoperative development of thyroid storm. Our observations imply that immediate operation should generally be considered for the emergency treatment of an imminent thyroid storm.

Adult↗

PubMed1993

Amphipathic alpha-helical structure does not predict the ability of receptor-derived synthetic peptides to interact with guanine nucleotide-binding regulatory proteins.

In guanine nucleotide-binding regulatory protein- (G protein) coupled receptors, an amphipathic alpha-helix has been postulated to be the common structural determinant in the NH2- and COOH-terminal portions of the third intracellular loop representing the major interaction site with the G proteins. Here we assessed the ability of six peptides derived from these sites of the human dopamine D1-, D2-, and beta 1-adrenergic receptors to either activate G proteins directly or to uncouple the activity of their respective receptors in a native membrane environment. In addition, the cross-reactivity was analyzed. Nonspecific effects occurring at high concentrations were differentiated from G protein-specific effects. The peptide D2N derived from the NH2-terminal part of the third intracellular loop of the dopamine D2 receptor was the only one capable of specifically reversing the action of its receptor, the dopamine-mediated inhibition of the adenylyl cyclase. Furthermore, only D2N stimulated pertussis toxin-sensitive G proteins. However, D2N as the only peptide exhibiting specific effects did not exhibit the predicted amphipathic alpha-helix observed for mastoparan (Higashijima, T., Burnier, J., and Ross, E. M. (1990) J. Biol. Chem. 265, 14176-14186) as demonstrated by circular dichroism spectroscopy. In contrast, a peptide for which a certain degree of helicality was verified spectroscopically (D1C) was neither active in GTPase and adenylyl cyclase determinations, nor did it block the receptor-mediated cyclase activation. Hence, the amphipathic alpha-helix does not represent the main structural determinant for the receptor-G protein interaction site.

Adenylyl Cyclases↗

PubMed1993

Stimulation of human thyroid growth via the inhibitory guanine nucleotide binding (G) protein Gi: constitutive expression of the G-protein alpha subunit Gi alpha-1 in autonomous adenoma.

The alpha subunits of the stimulatory and inhibitory G proteins, Gs alpha and Gi alpha, activate transmembrane-signaling systems involved in the control of cell proliferation. We have investigated the pattern of expression of Gi alpha subtypes and Gi alpha-mediated proliferative responses in the human thyroid. Human thyroid membranes contain two subtypes of Gi alpha, Gi alpha-1 and Gi alpha-2, as assessed by using specific antibodies. The expression of Gi alpha-1 is under tight control by thyrotropin in vivo and in primary cultures of thyroid epithelial cells. In contrast, Gi alpha-1 is expressed in the absence of thyrotropin in thyroid autonomous adenoma, an endocrine-active tumor, and its levels are not regulated by thyrotropin in thyroid epithelial cells prepared from these tumors. If thyroid epithelial cells are treated with pertussis toxin to block signal transduction via Gi, the mitogenic response to serum factors is reduced. These observations demonstrate that Gi subtypes transmit growth stimuli in the human thyroid. The constitutive expression of Gi alpha-1 in autonomous adenoma may allow for the unregulated stimulation of thyroid cell proliferation by a yet unidentified signaling pathway and, thus, be causally related to autonomous growth of thyroid cells.

Adenoma↗

PubMed1993

Binding and structure-activity-relation of benzo[f]isoquinoline- and norcodeinone-derivatives at mu-opioid receptors in the rat cerebral cortex.

1. We have probed the ligand binding site of the mu-opioid receptor using a series of isoquinoline- and norcodeinone-derivatives; in these morphine- and codeine-analogues, the position of the piperidine-nitrogen as well as its mobility is altered relative to that found in morphine. 2. The mu-receptor in rat cortical membranes was labelled with [3H]-naloxone and competition experiments were carried out in the absence and presence of Gpp(NH)p and NaCl: conditions, which are associated with affinity shifts for agonists whilst antagonist affinity remains unaffected. Moving the piperidine-nitrogen closer to the phenolic ring or reducing its mobility by incorporation into an additional ring drastically decreases the affinity. 3. In contrast, we find that the piperidine-nitrogen in a distal position is well tolerated provided that additional structural criteria, in particular a phenolic hydroxyl-group and a 6 carbon ring corresponding to ring C in morphine, are met. This assumption was verified by the synthesis of WB4/PH (4aR, 10bS, 11R)-10, 11-epoxy-1, 2, 3, 4, 5, 6-hexahydro-9-hydroxy-3-methyl-4a,10b-butano- benzo[f]isochinolin-12-on(10). This compound is an agonist with an affinity comparable to that of morphine. 4. We therefore conclude that both the mobility of the piperidine nitrogen of the ligand and of its counterpart anionic site in the ligand binding pocket of the mu-opioid receptor (presumably aspartic acid) are important determinants for fruitful interaction. The mobility of the anionic site is restricted in one direction but is sufficient to bridge the 2A distance that exists between the position of the nitrogen in morphine and WB4/PH.

Animals↗

PubMed1992

Phosphorylation of the recombinant spliced variants of the alpha-sub-unit of the stimulatory guanine-nucleotide binding regulatory protein (Gs) by the catalytic sub-unit of protein kinase A.

Both GS alpha-1 and GS alpha-4 were phosphorylated by the purified catalytic sub-unit of protein kinase A. Phosphate incorporation into 220 pmol and 190 pmol of GS alpha-4 and GS alpha-1 after a 1 hour incubation with kinase was 14 pmol and 10 pmol, respectively. These low levels of phosphorylation are due to the thermal lability of purified recombinant GS alpha. However, the phosphorylation was inhibited by guanine nucleotides (GDP-beta-S, GppNHp and GTP) and is, therefore, a specific event. We suggest that, as for GS alpha phosphorylation by protein kinase C (Pyne et al., 1992), the guanine nucleotide-free form of GS alpha is the most likely substrate. Guanine-nucleotides reduce the lifetime and, therefore availability for phosphorylation, of guanine-nucleotide free GS alpha. GS alpha phosphorylation by protein kinase A in vitro provides preliminary evidence that a similar phosphorylation of GS alpha may be an important regulatory event in cells.

Autoradiography↗

PubMed1992

Phosphorylation of the spliced variant forms of the recombinant stimulatory guanine-nucleotide-binding regulatory protein (Gs alpha) by protein kinase C.

Recombinant forms of Gs alpha-1 and Gs alpha-4 were shown to act as substrates for a purified preparation of brain protein kinase C. Both forms of Gs alpha were thermally denatured during the incubation such that phosphorylation was virtually complete (greater than 90%) after 30 min. The quantity of phosphate incorporated into approximately equivalent starting amounts of the two forms of Gs alpha (4.8 pmol of Gs alpha-1 and 5.5 pmol of Gs alpha-4) at maximal phosphorylation were 0.23 +/- 0.08 pmol for Gs alpha-1 and 0.56 +/- 0.12 pmol for Gs alpha-4. Since both forms of Gs alpha were thermally denatured to the same extent after 30 min, the increased phosphorylation state of Gs alpha-4 provides evidence that Gs alpha-4 contains an additional phosphorylation site. Bray and co-workers [Bray, Carter, Simmons, Guo, Puckett, Kamhollz, Spiegel & Nirenberg (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 8893-8897] proposed that an additional phosphorylation site may exist at the splice junction in Gs alpha-4. The guanine-nucleotide-free form of Gs alpha appears to be the preferred substrate for phosphorylation. This interpretation is based upon the following observations. (i) Guanosine 5'-[beta-thio]diphosphate at micromolar concentrations inhibits the susceptibility of Gs alpha to phosphorylation; (ii) beta gamma-subunits, which inhibit GDP release from Gs alpha-GDP at millimolar Mg2+ concentrations, also inhibit the susceptibility of Gs alpha to phosphorylation; and (iii) guanosine 5'[beta gamma-imido]triphosphate inhibits the susceptibility of Gs alpha to act as a substrate for phosphorylation. These studies suggest that there is potential for cross-talk between receptors which trigger PtdIns(4,5)P2 hydrolysis and subsequently protein kinase C activation, and receptors which stimulate adenylate cyclase via Gs.

Alkaloids↗

PubMed1992

Gi3 does not contribute to the inhibition of adenylate cyclase when stimulation of an alpha 2-adrenergic receptor causes activation of both Gi2 and Gi3.

Agonist occupancy of the alpha 2-C10 adrenergic receptor in a stable clone (1C) of Rat 1 fibroblasts produced by transfection of cells with genomic DNA encoding this receptor causes the activation of both of the pertussis-toxin-sensitive G-proteins Gi2 and Gi3 [Milligan, Carr, Gould, Mullaney & Lavan (1991) J. Biol. Chem. 266, 6447-6455]. An IgG fraction from an antiserum (I3B) which identifies the C-terminal decapeptide of Gi3 alpha only was able to inhibit partially receptor stimulation of high-affinity GTPase activity. An equivalent fraction from an antiserum (AS7) able to identify the C-terminal decapeptide of Gi1 alpha + Gi2 alpha, but not Gi3 alpha, was also able to inhibit partially receptor stimulation of GTPase activity, and the effects of the two antisera were additive. By contrast, agonist-mediated inhibition of forskolin-amplified adenylate cyclase activity was abolished completely by the IgG fraction of antiserum AS7, but was not decreased by treatment with antiserum 13B. Based on the proportion of agonist-stimulated high-affinity GTPase which was prevented by each antiserum and on the measured membrane levels of Gi2 and Gi3, calculations indicated that essentially all of the cellular Gi3, but only 15% of the available Gi2, can be activated by the alpha 2-C10 adrenergic receptor in these cells. These results demonstrate that, although Gi3 is activated by alpha 2-adrenergic agonists in membranes of clone 1C cells, it does not contribute to the transduction of receptor-mediated inhibition of adenylate cyclase.

Adenylyl Cyclase Inhibitors↗

PubMed1992

Functional expression of the human serotonin 5-HT1A receptor in Escherichia coli. Ligand binding properties and interaction with recombinant G protein alpha-subunits.

Signaling through serotonin 5-HT1A receptors involves multiple pathways. We have investigated the functional coupling of the human 5-HT1A receptor to different G proteins using an in vitro reconstitution system based on the expression of recombinant receptor (r5-HT1A) and G alpha-subunits (rG alpha) in Escherichia coli. The r5-HT1A receptor was expressed by insertion in a vector allowing its active expression in E. coli inner membranes. Binding of the selective agonist [3H] +/- 8-hydroxy-(2-N-dipropylamine)tetralin ([3H]8-OH-DPAT) to intact bacteria or E. coli membranes was saturable with a KD of approximately 8 nM and an average of 120 sites/bacterium. Binding properties of several serotoninergic ligands to r5-HT1A receptors were comparable with those measured in mammalian cells. Incubation of rG alpha.beta gamma with E. coli membranes resulted in high affinity agonist [3H]8-OH-DPAT binding (KD = 0.7 nM) and titration with a panel of rG alpha subtypes showed the order of potency: rGi alpha-3 greater than rGi alpha-2 greater than rGi alpha-1 much greater than rGo alpha, while rGs alpha appeared incapable of interacting with 5-HT1A receptors. Moreover, agonist-mediated enhancement of [35S]guanosine 5'-O-(3-thiotriphosphate) binding to rGi alpha confirmed the achievement of the functional interaction between receptor and G proteins. Our findings are in agreement with the in vivo ability of 5-HT1A receptors to activate Gi alpha related to adenylyl cyclase inhibition or K+ channel activation, but do not support previously reported adenylyl cyclase stimulation through interaction with Gs alpha.

8-Hydroxy-2-(di-n-propylamino)tetralin↗

PubMed1992

Reverse intrinsic activity of antagonists on G protein-coupled receptors.

Biological effects observed with an antagonist are usually interpreted as the result of its ability to block receptor activation produced by an endogenous agonist. In this Principles article, Wolfgang Schütz and Michael Freissmuth show how considerable evidence has now been accumulated for G protein-coupled receptors that antagonists not only bind to the receptor, but also induce a conformational change that favours uncoupling of the receptor from its G protein. The spontaneous activity of the unliganded receptor (i.e. the receptor not occupied by any ligand) is a well-established phenomenon in reconstituted systems with purified components. However, its physiological relevance needs to be verified in a more physiological environment before biological effect of antagonists can be primarily ascribed to negative intrinsic activity.

Animals↗