Publikationsserver
Voltage-dependence of adrenoceptor activity : Mechanisms and signal transduction
G protein-coupled receptors comprise a large superfamily of membrane proteins which transmit extracellular stimuli into intracellular responses. Adrenoceptors (AR) belong to the α-group of Rhodopsin-family G protein-coupled receptors. Physiological activation of adrenoceptors is mediated by binding...
Gespeichert in:
| 1. Verfasser: | |
|---|---|
| Beteiligte: | |
| Format: | Dissertation |
| Sprache: | Englisch |
| Veröffentlicht: |
Philipps-Universität Marburg
2015
|
| Schlagworte: | |
| Online Zugang: | PDF-Volltext |
| Tags: |
Tag hinzufügen
Keine Tags, Fügen Sie den ersten Tag hinzu!
|
| Zusammenfassung: | G protein-coupled receptors comprise a large superfamily of membrane proteins which transmit extracellular stimuli into intracellular responses. Adrenoceptors (AR) belong to the α-group of Rhodopsin-family G protein-coupled receptors. Physiological activation of adrenoceptors is mediated by binding of the endogenous catecholamines noradrenaline and adrenaline and leads to the regulation of neuronal, endocrine, cardiovascular, vegetative and metabolic functions. In addition to the classical activation of GPCRs by binding of ligands, experimental evidence has accumulated showing that GPCR activation can be modulated by the membrane potential. In this study adrenoceptors β1-AR and β2-AR have been investigated in regard to the modulation of their activity by the membrane potential. Both receptors couple to the stimulatory Gs protein. They were used as model receptors because despite increasing knowledge of voltage-dependent regulation of Gi- and Gq-coupled receptor activity, no data has been published on the voltage-sensitivity of Gs-coupled receptors.
Förster resonance energy transfer (FRET)-based assays were combined with whole-cell patch-clamp in a voltage-clamp mode in order to monitor agonist- and voltage-dependent alterations in either protein conformation or protein-protein interaction. Conformational changes of the receptor molecule upon stimulation with catecholamines in the presence or absence of depolarizing steps were directly monitored using FRET-based intramolecular receptor fusion proteins. By means of the combination of FRET and patch-clamp, voltage-dependence of β1-AR and to smaller extent β2-AR activation was shown to be an intrinsic property of these Gs-coupled receptors and was transmitted to downstream signaling. Receptor activity and downstream signaling decreased during depolarization. This expands voltage-dependent GPCRs to the group that couple to stimulatory G proteins. Voltage-dependence of β1-AR was mainly mediated via an alteration in efficacy of either Iso or Adr and occurred with highest sensitivity at physiological membrane potentials. Regarding Adr this is a unique finding since voltage-dependence of other GPCRs activated by their endogenous ligands were shown to rely on alterations in ligand affinity. Agonist-specificity of voltage-dependence was also apparent as dopamine-induced β1-AR interaction with arrestin 3 was enhanced during depolarization whereas it was reduced when induced by adrenaline, isoprenaline, noradrenaline or dobutamine. A different binding mode of dopamine might be responsible for these differences.
The second part of this thesis focused on the molecular mechanism underlying voltage-dependence. Mutation of a charged amino acid and a residue involved in agonist binding did not seem to alter voltage-dependence or only yielded inconclusive results. However, the introduction of an additional positive charge in the vicinity of a highly conserved aspartic acid in TM2 (D2.50) reduced voltage-dependence of the mutant receptor. We might have identified this aspartic acid as one residue of a voltage-sensor in α2A-AR but further experiments are required in order to corroborate this hypothesis.
Taken together these data expand voltage-dependent GPCRs to the group coupling to Gs proteins. Especially in excitable tissue like the heart, which expresses voltage-dependent β1-AR the membrane potential could fine-tune adrenoceptor signaling on a time scale within cardiac action potentials and in the presence of endogenous or synthetic catecholamines. On a molecular level we suggest a possible involvement of the highly conserved aspartic acid D2.50 in the detection of alterations in the membrane potential in α2A-AR. Whether this conclusion is also transferable to other G protein-coupled receptors remains to be investigated. |
|---|---|
| DOI: | 10.17192/z2016.0081 |