Biomedical subjects

R Jockers

Publications and source records attributed to R Jockers.

26 records · Page 2Linked to original sources

PubMed1996

New molecular and structural determinants involved in beta 2-adrenergic receptor desensitization and sequestration. Delineation using chimeric beta 3/beta 2-adrenergic receptors.

As the beta 3-adrenergic receptor (beta3AR) is resistant to short term agonist-promoted desensitization and sequestration, chimeric beta3/beta2 receptors were generated to identify the molecular determinants responsible for these regulatory processes in the beta2AR. By exchanging single or multiple intracellular domains of the beta3AR for the corresponding regions of the beta2AR, we show that specific domains can be identified as additive determinants for desensitization, while sequestration is more dependent on global structural conformation. The carboxyl-terminal tail, the third and the second intracellular loops of the beta2AR provided additive contributions to the desensitization observed upon short term agonist stimulation. The second intracellular loop plays a role which is as important as that of third cytoplasmic loop and carboxyl-terminal tail which had previously been identified as the major determinants of agonist-promoted desensitization. Additive contributions of the cytoplasmic domains of the beta2AR were also observed for agonist-promoted sequestration. The substitution of the first and second intracellular loops and the carboxyl tail were associated with a beta2-like sequestration phenotype. However, in contrast to what is observed for desensitization the co-substitution of the third cytoplasmic loop with any of the other domains completely suppressed sequestration. These results suggest that sequestration depends not only on appropriate interactions of multiple molecular determinants within the cytoplasmic region of the beta2AR but also on conformational determinants that may influence their orientation.

Adenylyl Cyclases↗

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

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↗

PubMed1993

Enhancement of immunoassay sensitivity by molecular modification of competitors.

The potential of weak competitors to enhance the sensitivity of competitive immunoassays is described. Several triazine derivatives have been analyzed for their use as competitors. Their binding properties were determined using an enzyme-linked immunosorbent assay (ELISA) with a monoclonal antibody specific to triazines. Selected derivatives were immobilized onto the surface of a fibre optic sensor and atrazine was determined in a competitive manner using a fluorescein-labelled antibody. Using the weak binding competitor 11-(4-ethylamino-6-methylthio-s-triazine-2-yl)undecanoic acid (TE11S) the detection limit for atrazine could be lowered 100-fold in comparison to 2-aminohexylamino-4-ethylamino-6-isopropylamino-s-triazine (AHA), the previously used competitor.

Antibodies, Monoclonal↗

PubMedDate not supplied

Interactions between aldehyde derivatives and the aldehyde binding site of bacterial luciferase.

The interaction of triazine aldehydes with the aldehyde binding site of bacterial luciferases was investigated using a series of triazine aldehydes with different aldehyde chain length, and substituents on the s-triazine ring. Substrate activity was determined using luciferase from Photobacterium fischeri and Vibrio harveyi in a dithionite-based luciferases assay. The chain length optimum was determined for two triazine aldehyde classes to be C-10 and C-11, respectively. Only the substrate activity of 10-(4-chloro-6-methylthio-s-triazine-2-yl)aminodecanal (5) was as high as n-decanal, the reference aldehyde. All other triazine derivatives reduced light emission, probably by hindered binding of the substrates. The degree of activity reduction correlated with the volume of the triazine ring moiety. The triazine moiety volume of compound 5 was estimated to be 200 x 10(-30) m3. Triazine aldehydes which showed reduced light emission had an estimated volume of 228 x 10(-30) m3 or greater. All triazine aldehydes showed approximately 10-fold lower activities for Vibrio harveyi than for Photobacterium fischeri luciferase. Substrate specificity was the same for both luciferases. A schematic superposition of quinone aldehydes and triazine aldehydes which showed substrate activities equivalent to n-decanal, indicated potential interaction sites of aldehyde substrates with the aldehyde binding site of bacterial luciferases. The in vivo relevance of the results is discussed.

Aldehydes↗

PubMedDate not supplied

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.

1-Methyl-3-isobutylxanthine↗

PubMedDate not supplied

Pineal and cortical melatonin receptors MT1 and MT2 are decreased in Alzheimer's disease.

The pineal hormone melatonin is involved in physiological transduction of temporal information from the light dark cycle to circadian and seasonal behavioural rhythms, as well as possessing neuroprotective properties. Melatonin and its receptors MT1 and MT2, which belong to the family of G protein-coupled receptors, are impaired in Alzheimer's disease (AD) with severe consequences to neuropathology and clinical symptoms. The present data provides the first immunohistochemical evidence for the cellular localization of the both melatonin receptors in the human pineal gland and occipital cortex, and demonstrates their alterations in AD. We localized MT1 and MT2 in the pineal gland and occipital cortex of 7 elderly controls and 11 AD patients using immunohistochemistry with peroxidase-staining. In the pineal gland both MT1 and MT2 were localized to pinealocytes, whereas in the cortex both receptors were expressed in some pyramidal and non-pyramidal cells. In patients with AD, parallel to degenerative tissue changes, there was an overall decrease in the intensity of receptors in both brain regions. In line with our previous findings, melatonin receptor expression in AD is impaired in two additional brain areas, and may contribute to disease pathology.

Aged↗

PubMedDate not supplied

[Signaling by melatonin receptors].

Melatonin is a hormone involved in various physiological processes such as the circadian cycle, hormone release and immune response. High-affinity melatonin receptors are classified in two pharmacologically distinct groups: Mel1 and Mel2. These receptors have first been localized in different organs and brain structures and some subtypes have since been cloned. Inhibition of adenylyl cyclase by Mel1 receptors is the best investigated signalling pathway but cannot be entirely responsible for the diversity of melatonin-induced phenomena. Phospholipase C, potassium ion channels and guanylyl cyclases have also been reported to be modulated by melatonin. This review updates present knowledge of the characterization and signalization of melatonin receptors.

Adenylyl Cyclase Inhibitors↗