Siluo Huang

Coupling of Agonist Binding to Effector Domain Activation in Metabotropic Glutamate-like Receptors

Many membrane receptors are made of a ligand binding domain and an effector domain mediating intracellular signaling. This is the case for the metabotropic glutamate-like G-protein-coupled receptors. How ligand binding leads to the active conformation of the effector domain in such receptors is largely unknown. Here, we used an evolutionary trace analysis and mutagenesis to identify critical residues involved in the allosteric coupling between the Venus flytrap ligand binding domain (VFT) and the heptahelical G-protein activating domain of the metabotropic glutamate-like receptors. We have shown that a conserved interdomain disulfide bridge is required for this allosteric interaction. Taking into account that these receptors are homodimers, this finding provides important new information explaining how the different conformations of the dimer of VFT lead to different signaling of such dimeric receptors.

Functioning of the dimeric GABAB receptor extracellular domain revealed by glycan wedge scanning

The G‐protein‐coupled receptor (GPCR) activated by the neurotransmitter GABA is made up of two subunits, GABAB1 and GABAB2. GABAB1 binds agonists, whereas GABAB2 is required for trafficking GABAB1 to the cell surface, increasing agonist affinity to GABAB1, and activating associated G proteins. These subunits each comprise two domains, a Venus flytrap domain (VFT) and a heptahelical transmembrane domain (7TM). How agonist binding to the GABAB1 VFT leads to GABAB2 7TM activation remains unknown. Here, we used a glycan wedge scanning approach to investigate how the GABAB VFT dimer controls receptor activity. We first identified the dimerization interface using a bioinformatics approach and then showed that introducing an N‐glycan at this interface prevents the association of the two subunits and abolishes all activities of GABAB2, including agonist activation of the G protein. We also identified a second region in the VFT where insertion of an N‐glycan does not prevent dimerization, but blocks agonist activation of the receptor. These data provide new insight into the function of this prototypical GPCR and demonstrate that a change in the dimerization interface is required for receptor activation.

Interdomain movements in metabotropic glutamate receptor activation

Many cell surface receptors are multimeric proteins, composed of several structural domains, some involved in ligand recognition, whereas others are responsible for signal transduction. In most cases, the mechanism of how ligand interaction in the extracellular domains leads to the activation of effector domains remains largely unknown. Here we examined how the extracellular ligand binding to the venus flytrap (VFT) domains of the dimeric metabotropic glutamate receptors activate the seven transmembrane (7TM) domains responsible for G protein activation. These two domains are interconnected by a cysteine-rich domain (CRD). We show that any of the four disulfide bridges of the CRD are required for the allosteric coupling between the VFT and the 7TM domains. More importantly, we show that a specific association of the two CRDs corresponds to the active state of the receptor. Indeed, a specific crosslinking of the CRDs with intersubunit disulfide bridges leads to fully constitutively active receptors, no longer activated by agonists nor by allosteric modulators. These data demonstrate that intersubunit movement at the level of the CRDs represents a key step in metabotropic glutamate receptor activation.

A New Family of Receptor Tyrosine Kinases with a Venus Flytrap Binding Domain in Insects and Other Invertebrates Activated by Aminoacids

Background Tyrosine kinase receptors (RTKs) comprise a large family of membrane receptors that regulate various cellular processes in cell biology of diverse organisms. We previously described an atypical RTK in the platyhelminth parasite Schistosoma mansoni, composed of an extracellular Venus flytrap module (VFT) linked through a single transmembrane domain to an intracellular tyrosine kinase domain similar to that of the insulin receptor. Methods and Findings Here we show that this receptor is a member of a new family of RTKs found in invertebrates, and particularly in insects. Sixteen new members of this family, named Venus Kinase Receptor (VKR), were identified in many insects. Structural and phylogenetic studies performed on VFT and TK domains showed that VKR sequences formed monophyletic groups, the VFT group being close to that of GABAB receptors and the TK one being close to that of insulin receptors. We show that a recombinant VKR is able to autophosphorylate on tyrosine residues, and report that it can be activated by L-arginine. This is in agreement with the high degree of conservation of the alpha amino acid binding residues found in many amino acid binding VFTs. The presence of high levels of vkr transcripts in larval forms and in female gonads indicates a putative function of VKR in reproduction and/or development. Conclusion The identification of RTKs specific for parasites and insect vectors raises new perspectives for the control of human parasitic and infectious diseases.

Evolution of the class C GPCR Venus flytrap modules involved positive selected functional divergence

BackgroundClass C G protein-coupled receptors (GPCRs) represent a distinct group of the GPCR family, which structurally possess a characteristically distinct extracellular domain inclusive of the Venus flytrap module (VFTM). The VFTMs of the class C GPCRs is responsible for ligand recognition and binding, and share sequence similarity with bacterial periplasmic amino acid binding proteins (PBPs). An extensive phylogenetic investigation of the VFTMs was conducted by analyzing for functional divergence and testing for positive selection for five typical groups of the class C GPCRs. The altered selective constraints were determined to identify the sites that had undergone functional divergence via positive selection. In order to structurally demonstrate the pattern changes during the evolutionary process, three-dimensional (3D) structures of the GPCR VFTMs were modelled and reconstructed from ancestral VFTMs.ResultsOur results show that the altered selective constraints in the VFTMs of class C GPCRs are statistically significant. This implies that functional divergence played a key role in characterizing the functions of the VFTMs after gene duplication events. Meanwhile, positive selection is involved in the evolutionary process and drove the functional divergence of the VFTMs. Our results also reveal that three continuous duplication events occurred in order to shape the evolutionary topology of class C GPCRs. The five groups of the class C GPCRs have essentially different sites involved in functional divergence, which would have shaped the specific structures and functions of the VFTMs.ConclusionTaken together, our results show that functional divergence involved positive selection and is partially responsible for the evolutionary patterns of the class C GPCR VFTMs. The sites involved in functional divergence will provide more clues and candidates for further research on structural-function relationships of these modules as well as shedding light on the activation mechanism of the class C GPCRs.

GABAB receptor subunit GB1 at the cell surface independently activates ERK1/2 through IGF-1R transactivation.

Background Functional GABAB receptor is believed to require hetero-dimerization between GABAB1 (GB1) and GABAB2 (GB2) subunits. The GB1 extracellular domain is required for ligand binding, and the GB2 trans-membrane domain is responsible for coupling to G proteins. Atypical GABAB receptor responses observed in GB2-deficient mice suggested that GB1 may have activity in the absence of GB2. However the underlying mechanisms remain poorly characterized. Methodology/Principal Findings Here, by using cells overexpressing a GB1 mutant (GB1asa) with the ability to translocate to the cell surface in the absence of GB2, we show that GABAB receptor agonists, such as GABA and Baclofen, can induce ERK1/2 phosphorylation in the absence of GB2. Furthermore, we demonstrate that GB1asa induces ERK1/2 phosphorylation through Gi/o proteins and PLC dependent IGF-1R transactivation. Conclusions/Significance Our data suggest that GB1 may form a functional receptor at the cell surface in the absence of GB2.

GABAB receptor promotes its own surface expression by recruiting a Rap1-dependent signaling cascade

ABSTRACT G-protein-coupled receptors (GPCRs) are key players in cell signaling, and their cell surface expression is tightly regulated. For many GPCRs such as β2-AR (β2-adrenergic receptor), receptor activation leads to downregulation of receptor surface expression, a phenomenon that has been extensively characterized. By contrast, some other GPCRs, such as GABAB receptor, remain relatively stable at the cell surface even after prolonged agonist treatment; however, the underlying mechanisms are unclear. Here, we identify the small GTPase Rap1 as a key regulator for promoting GABAB receptor surface expression. Agonist stimulation of GABAB receptor signals through Gαi/o to inhibit Rap1GAPII (also known as Rap1GAP1b, an isoform of Rap1GAP1), thereby activating Rap1 (which has two isoforms, Rap1a and Rap1b) in cultured cerebellar granule neurons (CGNs). The active form of Rap1 is then recruited to GABAB receptor through physical interactions in CGNs. This Rap1-dependent signaling cascade promotes GABAB receptor surface expression by stimulating receptor recycling. Our results uncover a new mechanism regulating GPCR surface expression and also provide a potential explanation for the slow, long-lasting inhibitory action of GABA neurotransmitter. Summary: GABAB receptor activation induces Rap1 activation to promote its own surface expression by stimulating receptor recycling, thereby providing a potential explanation for the long-lasting action of GABA.

Allosteric control of an asymmetric transduction in a G protein-coupled receptor heterodimer

GPCRs play critical roles in cell communication. Although GPCRs can form heteromers, their role in signaling remains elusive. Here we used rat metabotropic glutamate (mGlu) receptors as prototypical dimers to study the functional interaction between each subunit. mGluRs can form both constitutive homo- and heterodimers. Whereas both mGlu2 and mGlu4 couple to G proteins, G protein activation is mediated by mGlu4 heptahelical domain (HD) exclusively in mGlu2-4 heterodimers. Such asymmetric transduction results from the action of both the dimeric extracellular domain, and an allosteric activation by the partially-activated non-functional mGlu2 HD. G proteins activation by mGlu2 HD occurs if either the mGlu2 HD is occupied by a positive allosteric modulator or if mGlu4 HD is inhibited by a negative modulator. These data revealed an oriented asymmetry in mGlu heterodimers that can be controlled with allosteric modulators. They provide new insight on the allosteric interaction between subunits in a GPCR dimer.

The GluN1/GluN2B NMDA receptor and metabotropic glutamate receptor 1 negative allosteric modulator has enhanced neuroprotection in a rat subarachnoid hemorrhage model

ABSTRACT Excessive glutamate in cerebrospinal fluid after subarachnoid hemorrhage (SAH) causes excitotoxic damage through calcium overloading and a subsequent apoptotic cascade. GluN1/GluN2B containing N‐methyl‐Daspartate (NMDA) receptor and metabotropic glutamate receptor 1 (mGluR1) can play a leading role in glutamate‐mediated excitotoxicity. Here we report that Ifenprodil (100 &mgr;M), a negative allosteric modulator (NAM) of GluN1/GluN2B NMDA receptors, and JNJ16259685 (10 &mgr;M), a NAM of mGluR1, have an additive efficacy against glutamate (100 &mgr;M)‐induced Ca2+ release and cell apoptosis in primary cortical, hippocampal, and cerebellar granule neurons. Compared with intraperitoneal injection of Ifenprodil (10 mg/kg) and JNJ16259685 (1 mg/kg) separately, the combination therapy of Ifenprodil plus JNJ16259685 significantly improves the neurological deficit at 24 h and 72 h after experimental SAH. It reduces the number of TUNEL/DAPI‐positive and activated caspase‐3/NeuN‐positive cells in cortical and hippocampal CA1 regions at 72 h, decreases levels of glutamate in cerebrospinal fluid at 72 h, and reduces the mitochondrial Ca2+ concentration. Meanwhile, the combination therapy attenuates apoptosis as shown by an increased Bcl‐2 expression, decreased Bax expression and release of cytochrome c, and reduction of cleaved caspase‐9 and caspase‐3 at 24 h after SAH. These findings indicate that targeting both the intracellular Ca2+ overloading and neuronal apoptosis using the Ifenprodil and JNJ16259685 is a promising new therapy for SAH. Graphical abstract Excessive glutamate causes over‐stimulation of GluN1/GluN2B NMDA receptors and mGluR1, which in turn contributes to the calcium overload and cell death. Blockage of GluN1/GluN2B NMDA receptors and mGluR1 using combination therapy of Ifenprodil and JNJ16259685 has additive efficacy against SAH‐induced neurological deficit and neuronal apoptosis. Figure. No Caption available. HighlightsIfenprodil and JNJ16259685 have additive effect against glutamate‐mediated Ca2+ overloading and cell apoptosis.Ifenprodil and JNJ16259685 have enhanced neuroprotection against early brain injury after SAH.A dual‐target inhibition of Ca2+ overloading and apoptosis using the Ifenprodil and JNJ16259685