Evidence for activity-regulated hormone-binding cooperativity across glycoprotein hormone receptor homomers.

Glycoprotein hormone receptors show strong negative cooperativity. As a consequence, at physiological hormone concentrations, a single agonist binds to a receptor dimer. Here we present evidence that constitutively active receptors lose cooperative allosteric regulation in direct relation with their basal activity.

Deletion of the RNaseIII enzyme dicer in thyroid follicular cells causes hypothyroidism with signs of neoplastic alterations.

Micro-RNAs (miRNAs) are small non-coding RNAs that regulate gene expression, mainly at mRNA post-transcriptional level. Functional maturation of most miRNAs requires processing of the primary transcript by Dicer, an RNaseIII-type enzyme. To date, the importance of miRNA function for normal organogenesis has been demonstrated in several mouse models of tissue-specific Dicer inactivation.

Detection of antigen interactions ex vivo by proximity ligation assay: endogenous dopamine D2-adenosine A2A receptor complexes in the striatum.

The existence of G protein-coupled receptor (GPCR) dimers and/or oligomers has been demonstrated in heterologous systems using a variety of biochemical and biophysical assays. While these interactions are the subject of intense research because of their potential role in modulating signaling and altering pharmacology, evidence for the existence of receptor interactions in vivo is still elusive because of a lack of appropriate methods to detect them. Here, we adapted and optimized a proximity ligation assay (PLA) for the detection in brain slices of molecular proximity of two antigens located on either the same or two different GPCRs.

CODA-RET reveals functional selectivity as a result of GPCR heteromerization.

Here we present a new method that combines protein complementation with resonance energy transfer to study conformational changes in response to activation of a defined G protein-coupled receptor heteromer, and we apply the approach to the putative dopamine D1-D2 receptor heteromer. Remarkably, the potency of the D2 dopamine receptor (D2R) agonist R-(-)-10,11-dihydroxy-N-n-propylnoraporphine (NPA) to change the Gα(i) conformation via the D2R protomer in the D1-D2 heteromer was enhanced ten-fold relative to its potency in the D2R homomer. In contrast, the potencies of the D2R agonists dopamine and quinpirole were the same in the homomer and heteromer.

Making structural sense of dimerization interfaces of delta opioid receptor homodimers.

Opioid receptors, like other members of the G protein-coupled receptor (GPCR) family, have been shown to associate to form dimers and/or oligomers at the plasma membrane. Whether this association is stable or transient is not known. Recent compelling evidence suggests that at least some GPCRs rapidly associate and dissociate.

G protein-coupled receptor kinase-2 constitutively regulates D2 dopamine receptor expression and signaling independently of receptor phosphorylation.

We investigated the regulatory effects of GRK2 on D(2) dopamine receptor signaling and found that this kinase inhibits both receptor expression and functional signaling in a phosphorylation-independent manner, apparently through different mechanisms. Overexpression of GRK2 was found to suppress receptor expression at the cell surface and enhance agonist-induced internalization, whereas short interfering RNA knockdown of endogenous GRK2 led to an increase in cell surface receptor expression and decreased agonist-mediated endocytosis. These effects were not due to GRK2-mediated phosphorylation of the D(2) receptor as a phosphorylation-null receptor mutant was regulated similarly, and overexpression of a catalytically inactive mutant of GRK2 produced the same effects.

Hetero-oligomerization of CCR2, CCR5, and CXCR4 and the protean effects of "selective" antagonists.

Chemokine receptors constitute an attractive family of drug targets in the frame of inflammatory diseases. However, targeting specific chemokine receptors may be complicated by their ability to form dimers or higher order oligomers. Using a combination of luminescence complementation and bioluminescence resonance energy transfer assays, we demonstrate for the first time the existence of hetero-oligomeric complexes composed of at least three chemokine receptors (CCR2, CCR5, and CXCR4).

Allosteric communication between protomers of dopamine class A GPCR dimers modulates activation.

A major obstacle to understanding the functional importance of dimerization between class A G protein-coupled receptors (GPCRs) has been the methodological limitation in achieving control of the identity of the components comprising the signaling unit. We have developed a functional complementation assay that enables such control, and we demonstrate it here for the human dopamine D2 receptor. The minimal signaling unit, two receptors and a single G protein, is maximally activated by agonist binding to a single protomer, which suggests an asymmetrical activated dimer.

Dopamine D2 receptors form higher order oligomers at physiological expression levels.

G-protein-coupled receptors are generally thought to be organized as dimers; whether they form higher order oligomers is a topic of much controversy. We combined bioluminescence/fluorescence complementation and energy transfer to demonstrate that at least four dopamine D2 receptors are located in close molecular proximity in living mammalian cells, consistent with their organization as higher order oligomers at the plasma membrane. This implies the existence of multiple receptor interfaces.

Recruitment of beta-arrestin2 to the dopamine D2 receptor: insights into anti-psychotic and anti-parkinsonian drug receptor signaling.

Drugs acting at dopamine D2-like receptors play a pivotal role in the treatment of both schizophrenia and Parkinson's disease. Recent studies have demonstrated a role for G-protein independent D2 receptor signaling pathways acting through beta-arrestin. In this study we describe the establishment of a Bioluminescence Resonance Energy Transfer (BRET) assay for measuring dopamine induced recruitment of human beta-arrestin2 to the human dopamine D2 receptor.

Allosteric properties of G protein-coupled receptor oligomers.

Allosteric regulation of ligand binding is a well-established mechanism regulating the function of G protein-coupled receptors (GPCR). Allosteric modulators have been considered so far as molecules binding to an allosteric site, distinct from that of the reference ligand (orthosteric site), and able to modulate the binding affinity at the orthosteric site and/or the signaling properties resulting from orthosteric site occupancy. Given that most GPCR are known to form dimers or higher order oligomers, we explored whether allosteric interactions could also occur between protomers within oligomeric arrays, thereby influencing binding and signaling receptor properties.

F2L, a peptide derived from heme-binding protein, chemoattracts mouse neutrophils by specifically activating Fpr2, the low-affinity N-formylpeptide receptor.

F2L (formylpeptide receptor (FPR)-like (FPRL)-2 ligand), a highly conserved acetylated peptide derived from the amino-terminal cleavage of heme-binding protein, is a potent chemoattractant for human monocytes and dendritic cells, and inhibits LPS-induced human dendritic cell maturation. We recently reported that F2L is able to activate the human receptors FPRL-1 and FPRL2, two members of the FPR family, with highest selectivity and affinity for FPRL2. To facilitate delineation of mechanisms of F2L action in vivo, we have now attempted to define its mouse receptors.

Allosteric modulation of binding properties between units of chemokine receptor homo- and hetero-oligomers.

We have demonstrated previously that the chemokine receptors CCR2 and CCR5 form homo- and heterodimers and that dimers can only bind a single chemokine molecule with high affinity. We provide here evidence from bioluminescence resonance energy transfer experiments that stimulation by chemokines does not influence the CCR2/CCR5 heterodimerization status. In addition, we show that the rate of radioligand dissociation from one unit of the heterodimer in "infinite" tracer dilution conditions is strongly increased in the presence of an unlabeled chemokine ligand of the other unit.

Specificity and promiscuity of gonadotropin receptors.

The dichotomy between hormone recognition by the ectodomain and activation of the G protein by the rhodopsin-like serpentine portion is a well established property of glycoprotein hormone receptors. The specificity barrier avoiding promiscuous activation of the FSH receptor by the high concentration of human chorionic gonadotropin (hCG) prevailing during human pregnancy was thus believed to lie in the ectodomain. In the past two years, mutations responsible for rare spontaneous cases of ovarian hyperstimulation syndromes have partially modified this simple view.

Dimerization of chemokine receptors and its functional consequences.

It became clear over the recent years that most, if not all, G protein-coupled receptors (GPCR) are able to form dimers or higher order oligomers. Chemokine receptors make no exception to this new rule and both homo- and heterodimerization were demonstrated for CC and CXC receptors. Functional analyses demonstrated negative binding cooperativity between the two subunits of a dimer.

Glycoprotein hormone receptors: link between receptor homodimerization and negative cooperativity.

The monomeric model of rhodopsin-like G protein-coupled receptors (GPCRs) has progressively yielded the floor to the concept of GPCRs being oligo(di)mers, but the functional correlates of dimerization remain unclear. In this report, dimers of glycoprotein hormone receptors were demonstrated in living cells, with a combination of biophysical (bioluminescence resonance energy transfer and homogenous time resolved fluorescence/fluorescence resonance energy transfer), functional and biochemical approaches. Thyrotropin (TSHr) and lutropin (LH/CGr) receptors form homo- and heterodimers, via interactions involving primarily their heptahelical domains.

An activation switch in the rhodopsin family of G protein-coupled receptors: the thyrotropin receptor.

We aimed at understanding molecular events involved in the activation of a member of the G protein-coupled receptor family, the thyrotropin receptor. We have focused on the transmembrane region and in particular on a network of polar interactions between highly conserved residues. Using molecular dynamics simulations and site-directed mutagenesis techniques we have identified residue Asn-7.