G protein-coupled receptors are known to form homo- and heteromers at the plasma membrane, but the stoichiometry of these receptor oligomers are relatively unknown. Here, by using bimolecular fluorescence complementation, we visualized for the first time the occurrence of heterodimers of metabotropic glutamate mGlu5 receptors (mGlu5R) and dopamine D2 receptors (D2R) in living cells. Furthermore, the combination of bimolecular fluorescence complementation and bioluminescence resonance energy transfer techniques, as well as the sequential resonance energy transfer (SRET) technique, allowed us to detect the occurrence receptor oligomers containing more than two protomers, mGlu5R, D2R and adenosine A2A receptor (A2AR). Interestingly, by using high-resolution immunoelectron microscopy we could confirm that the three receptors co-distribute within the extrasynaptic plasma membrane of the same dendritic spines of asymmetrical, putative glutamatergic, striatal synapses. Also, co-immunoprecipitation experiments in native tissue demonstrated the existence of an association of mGlu5R, D2R and A2AR in rat striatum homogenates. Overall, these results provide new insights into the molecular composition of G protein-coupled receptor oligomers in general and the mGlu5R/D2R/A2AR oligomer in particular, a receptor oligomer that might constitute an important target for the treatment of some neuropsychiatric disorders.
The results presented in this paper show that adenosine A 2A receptor (A 2A R) form homodimers and that homodimers but not monomers are the functional species at the cell surface. Fluorescence resonance energy transfer (FRET) and bioluminescence resonance energy transfer (BRET) techniques have been used to demonstrate in transfected HEK293 cells homodimerization of A 2A R, which are heptaspanning membrane receptors with enriched expression in striatum. The existence of homodimers at the cell surface was demonstrated by time-resolved FRET. Although agonist activation of the receptor leads to the formation of receptor clusters, it did not affect the degree of A 2A R-A 2A R dimerization. Both monomers and dimers were detected by immunoblotting in cell extracts. However, cell surface biotinylation of proteins has made evident that more than 90% of the cell surface receptor is in its dimeric form. Thus, it seems that homodimers are the functional form of the receptor present on the plasma membrane.A deletion mutant version of the A 2A receptor, lacking its C-terminal domain, was also able to form both monomeric and dimeric species when cell extracts from transfected cells were analyzed by immunoblotting. This suggests that the C-terminal tail does not participate in the dimerization. This is relevant as the C-terminal tail of A 2A R is involved in heteromers formed by A 2A R and dopamine D2 receptors. BRET ratios corresponding to A 2A R-A 2A R homodimers were higher than those encountered for heterodimers formed by A 2A R and dopamine D2 receptors. As A 2A R and dopamine D2 receptors do indeed interact, these results indicate that A 2A R homodimers are the functional species at the cell surface and that they coexist with A 2A R/D2 receptor heterodimers. Keywords: 7TM receptors, dopamine receptors, G proteincoupled receptors, heteromerization, homomerization, receptor-receptor interactions. Heptaspanning membrane receptors (HSMRs) also known as G protein-coupled receptors (GPCRs) were previously considered to be monomeric proteins which only interacted with G proteins. However it has become clear that HSMRs are oligomeric structures formed by receptor homodimers, heterodimers and multimers and a variety of proteins interacting at the horizontal and the vertical level (Agnati et al. 2002Bouvier, 2001;Milligan and White 2001;Rios et al. 2001;Franco et al. 2003
Hutchinson-Gilford progeria syndrome (HGPS) is a rare genetic disease caused by defective prelamin A processing, leading to nuclear lamina alterations, severe cardiovascular pathology, and premature death. Prelamin A alterations also occur in physiological aging. It remains unknown how defective prelamin A processing affects the cardiac rhythm. We show age-dependent cardiac repolarization abnormalities in HGPS patients that are also present in the Zmpste24−/− mouse model of HGPS. Challenge of Zmpste24−/− mice with the β-adrenergic agonist isoproterenol did not trigger ventricular arrhythmia but caused bradycardia-related premature ventricular complexes and slow-rate polymorphic ventricular rhythms during recovery. Patch-clamping in Zmpste24 −/− cardiomyocytes revealed prolonged calcium-transient duration and reduced sarcoplasmic reticulum calcium loading and release, consistent with the absence of isoproterenol-induced ventricular arrhythmia. Zmpste24 −/− progeroid mice also developed severe fibrosis-unrelated bradycardia and PQ interval and QRS complex prolongation. These conduction defects were accompanied by overt mislocalization of the gap junction protein connexin43 (Cx43). Remarkably, Cx43 mislocalization was also evident in autopsied left ventricle tissue from HGPS patients, suggesting intercellular connectivity alterations at late stages of the disease. The similarities between HGPS patients and progeroid mice reported here strongly suggest that defective cardiac repolarization and cardiomyocyte connectivity are important abnormalities in the HGPS pathogenesis that increase the risk of arrhythmia and premature death.Hutchinson-Gilford progeria syndrome | progerin | prelamin A | connexin43 | calcium handling T he LMNA gene encodes A-type lamins (lamin A and lamin C), key components of the mammalian nuclear envelope with important structural and regulatory functions that affect signaling, transcription, and chromatin organization among other processes (1). Mature lamin A is produced from the precursor prelamin A through a series of posttranslational modifications, consisting of sequential farnesylation at the cysteine in the Cysteine-SerineIsoleucine-Methionine motif, cleavage of the Serine-IsoleucineMethionine residues, carboxymethylation of the newly accessible cysteine, and a final proteolytic cleavage by the zinc metallopeptidase STE24 (ZMPSTE24, also called FACE-1) (2).Mutations in the human LMNA gene or defective processing of prelamin A cause a group of diseases termed laminopathies, including the premature aging disorder Hutchinson-Gilford progeria syndrome (HGPS), a very rare genetic disorder with an estimated prevalence of 1 in 21 million people (www.progeriaresearch.org). SignificanceDefective prelamin A processing causes cardiovascular alterations and premature death in Hutchinson-Gilford progeria syndrome (HGPS) patients and also occurs during physiological aging. We found overt repolarization abnormalities in HGPS patients at advanced disease stages. Similar alterations were present in proger...
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