Interdomain interaction of Stat3 regulates its Src homology 2 domain-mediated receptor binding activity

Activation of Stat proteins by cytokines is initiated by their Src homology 2 (SH2) domain-mediated association with the cytokine receptors. Previously, we identified an essential role of the coiled-coil domain of Stat3 in binding of the receptor peptides derived from the interleukin-6 receptor subunit, gp130. In this study, we further investigated the molecular basis of this regulation. We found that the C-terminal domain of Stat3 negatively regulates its receptor binding activity only in the absence of the first alpha-helix of the coiled-coil domain, which leads to a hypothesis of intramolecular interaction. Physical interactions between the coiled-coil domain and the C-terminal domain, as well as the SH2 domain, were indeed detected. Furthermore, a sub-region of the C-terminal domain (amino acids 720-740), which is also involved in the interaction with the coiled-coil domain, was demonstrated to be critical for the regulation of the receptor binding. Correspondingly, phosphorylation on Ser-727 within this region inhibits this interaction. In agreement with the peptide binding results, both the coiled-coil domain and the C-terminal sub-region are necessary for the functional recruitment of Stat3 to the cellular gp130 in response to interleukin-6, suggesting that the interdomain interaction is a prerequisite for the SH2-mediated receptor binding in interleukin-6 signaling.     

Functional importance of the coiled-coil of the Ebola virus glycoprotein

Ebola virus contains a single glycoprotein (GP) that is responsible for receptor binding and membrane fusion and is proteolytically cleaved into disulfide-linked GP1 and GP2 subunits. The GP2 subunit possesses a coiled-coil motif, which plays an important role in the oligomerization and fusion activity of other viral GPs. To determine the functional significance of the coiled-coil motif of GP2, we examined the effects of peptides corresponding to the coiled-coil motif of GP2 on the infectivity of a mutant vesicular stomatitis virus (lacking the receptor-binding/fusion protein) pseudotyped with the Ebola virus GP. A peptide corresponding to the C-terminal helix reduced the infectivity of the pseudotyped virus. We next introduced alanine substitutions into hydrophobic residues in the coiled-coil motif to identify residues important for GP function. None of the substitutions affected GP oligomerization, but some mutations, two in the N-terminal helix and all in the C-terminal helix, reduced the ability of GP to confer infectivity to the mutant vesicular stomatitis virus without affecting the transport of GP to the cell surface, its incorporation into virions, and the production of virus particles. These results indicate that the coiled-coil motif of GP2 plays an important role in facilitating the entry of Ebola virus into host cells and that peptides corresponding to this region could act as efficient antiviral agents.     

A trafficking checkpoint controls GABA(B) receptor heterodimerization

Surface expression of GABA(B) receptors requires heterodimerization of GB1 and GB2 subunits, but little is known about mechanisms that ensure efficient heterodimer assembly. We found that expression of the GB1 subunit on the cell surface is prevented through a C-terminal retention motif RXR(R); this sequence is reminiscent of the ER retention/retrieval motif RKR identified in subunits of the ATP-sensitive K+ channel. Interaction of GB1 and GB2 through their C-terminal coiled-coil alpha helices masks the retention signal in GB1, allowing the plasma membrane expression of the assembled complexes. Because individual GABA(B) receptor subunits and improperly assembled receptor complexes are not functional even if expressed on the cell surface, we conclude that a trafficking checkpoint ensures efficient assembly of functional GABA(B) receptors.     

Lateral diffusion of the GABAB receptor is regulated by the GABAB2 C terminus

GABAB (gamma-aminobutyric acid, type B) is a heterodimeric G-protein-coupled receptor. The GABAB1 subunit, which contains an endoplasmic reticulum retention sequence, is only transported to the cell surface when it is associated with the GABAB2 subunit. Fluorescence recovery after photobleaching studies in transfected COS-7 cells and hippocampal neurons revealed that GABAB2 diffuses slowly within the plasma membrane whether expressed alone or with the GABAB1 subunit. Treatment of cells with brefeldin A revealed that GABAB2 moves freely within the endoplasmic reticulum, suggesting that slow movement of GABAB2 is a result of its plasma membrane insertion. Disruption of the cytoskeleton did not affect the mobility of GABAB2, indicating that its restricted diffusion is not due to direct interactions with actin or tubulin. To determine whether the C terminus of GABAB2 regulates its diffusion, this region of the subunit was attached to the lymphocyte membrane protein, CD2, which then exhibited a slower rate of lateral diffusion. Furthermore, co-expression of a cytoplasmically expressed soluble form of the GABAB2 C terminus increased movement of the GABAB2 subunit. We constructed forms of GABAB2 with various C-terminal truncations. Truncation of GABAB2 after residue 862, but not residue 886, caused a dramatic increase in its mobility, suggesting that the region between these two residues is critical for restricting GABAB2 diffusion. Finally, we investigated whether activation of GABAB might modulate its movement. Treatment of COS-7 cells with the GABAB receptor agonist baclofen significantly increased its mobile fraction. These data show that the restricted movement of GABAB at the cell surface is regulated by a region within its C terminus.     

Trans-activation between 7TM domains: implication in heterodimeric GABAB receptor activation

Seven-transmembrane domain (7TM) receptors have important functions in cell-cell communication and can assemble into dimers or oligomers. Such complexes may allow specific functional cross-talk through trans-activation of interacting 7TMs, but this hypothesis requires further validation. Herein, we used the GABAB receptor, which is composed of two distinct subunits, GABAB1, which binds the agonist, and GABAB2, which activates G proteins, as a model system. By using a novel orthogonal-labelling approach compatible with time-resolved FRET and based on ACP- and SNAP-tag technologies to verify the heterodimerization of wild-type and mutated GABAB subunits, we demonstrate the existence of a direct allosteric coupling between the 7TMs of GABAB heterodimers. Indeed, a GABAB receptor, in which the GABAB2 extracellular domain was deleted, was still capable of activating G proteins. Furthermore, synthetic ligands for the GABAB2 7TM could increase agonist affinity at the GABAB1 subunit in this mutated receptor. In addition to bringing new information on GABAB receptor activation, these data clearly demonstrate the existence of direct trans-activation between the 7TM of two interacting proteins.     

Molecular determinants involved in the allosteric control of agonist affinity in the GABAB receptor by the GABAB2 subunit

The gamma-aminobutyric acid type B (GABAB) receptor is an allosteric complex made of two subunits, GABAB1 (GB1) and GABAB2 (GB2). Both subunits are composed of an extracellular Venus flytrap domain (VFT) and a heptahelical domain (HD). GB1 binds GABA, and GB2 plays a major role in G-protein activation as well as in the high agonist affinity state of GB1. How agonist affinity in GB1 is regulated in the receptor remains unknown. Here, we demonstrate that GB2 VFT is a major molecular determinant involved in this control. We show that isolated versions of GB1 and GB2 VFTs in the absence of the HD and C-terminal tail can form hetero-oligomers as shown by time-resolved fluorescence resonance energy transfer (based on HTRF technology). GB2 VFT and its association with GB1 VFT controlled agonist affinity in GB1 in two ways. First, GB2 VFT exerted a direct action on GB1 VFT, as it slightly increased agonist affinity in isolated GB1 VFT. Second and most importantly, GB2 VFT prevented inhibitory interaction between the two main domains (VFT and HD) of GB1. According to this model, we propose that GB1 HD prevents the possible natural closure of GB1 VFT. In contrast, GB2 VFT facilitates this closure. Finally, such inhibitory contacts between HD and VFT in GB1 could be similar to those important to maintain the inactive state of the receptor.     

Structural domains required for channel function of the mouse transient receptor potential protein homologue TRP1beta

Transient receptor potential proteins (TRP) are supposed to participate in the formation of store-operated Ca(2+) influx channels by co-assembly. However, little is known which domains facilitate the interaction of subunits. Contribution of the N-terminal coiled-coil domain and ankyrin-like repeats and the putative pore region of the mouse TRP1beta (mTRP1beta) variant to the formation of functional cation channels were analyzed following overexpression in HEK293 (human embryonic kidney) cells. MTRP1beta expressing cells exhibited enhanced Ca(2+) influx and enhanced whole-cell membrane currents compared to mTRP1beta deletion mutants. Using a yeast two-hybrid assay only the coiled-coil domain facilitated homodimerization of the N-terminus. These results suggest that the N-terminus of mTRP1beta is required for structural organization thus forming functional channels.     

Binding of the delta subunit to rod phosphodiesterase catalytic subunits requires methylated, prenylated C-termini of the catalytic subunits

PDE6 (type 6 phosphodiesterase) from rod outer segments consists of two types of catalytic subunits, alpha and beta; two inhibitory gamma subunits; and one or more delta subunits found only on the soluble form of the enzyme. About 70% of the phosphodiesterase activity found in rod outer segments is membrane-bound, and is thought to be anchored to the membrane through C-terminal prenyl groups. The recombinant delta subunit has been shown to solubilize the membrane-bound form of the enzyme. This paper describes the site and mechanism of this interaction in more detail. In isolated rod outer segments, the delta subunit was found exclusively in the soluble fraction, and about 30% of it did not coimmunoprecipitate with the catalytic subunits. The delta subunit that was bound to the catalytic subunits dissociated slowly, with a half-life of about 3.5 h. To determine whether the site of this strong binding was the C-termini of the phosphodiesterase catalytic subunits, peptides corresponding to the C-terminal ends of the alpha and beta subunits were synthesized. Micromolar concentrations of these peptides blocked the phosphodiesterase/delta subunit interaction. Interestingly, this blockade only occurred if the peptides were both prenylated and methylated. These results suggested that a major site of interaction of the delta subunit is the methylated, prenylated C-terminus of the phosphodiesterase catalytic subunits. To determine whether the catalytic subunits of the full-length enzyme are methylated in situ when bound to the delta subunit, we labeled rod outer segments with a tritiated methyl donor. Soluble phosphodiesterase from these rod outer segments was more highly methylated (4.5 +/- 0.3-fold) than the membrane-bound phosphodiesterase, suggesting that the delta subunit bound preferentially to the methylated enzyme in the outer segment. Together these results suggest that the delta subunit/phosphodiesterase catalytic subunit interaction may be regulated by the C-terminal methylation of the catalytic subunits.     

Gamma-aminobutyric acid type B (GABA(B)) receptor internalization is regulated by the R2 subunit

γ-Aminobutyric acid type B (GABA(B)) receptors are important for slow synaptic inhibition in the CNS. The efficacy of inhibition is directly related to the stability of cell surface receptors. For GABA(B) receptors, heterodimerization between R1 and R2 subunits is critical for cell surface expression and signaling, but how this determines the rate and extent of receptor internalization is unknown. Here, we insert a high affinity α-bungarotoxin binding site into the N terminus of the R2 subunit and reveal its dominant role in regulating the internalization of GABA(B) receptors in live cells. To simultaneously study R1a and R2 trafficking, a new α-bungarotoxin binding site-labeling technique was used, allowing α-bungarotoxin conjugated to different fluorophores to selectively label R1a and R2 subunits. This approach demonstrated that R1a and R2 are internalized as dimers. In heterologous expression systems and neurons, the rates and extents of internalization for R1aR2 heteromers and R2 homomers are similar, suggesting a regulatory role for R2 in determining cell surface receptor stability. The fast internalization rate of R1a, which has been engineered to exit the endoplasmic reticulum, was slowed to that of R2 by truncating the R1a C-terminal tail or by removing a dileucine motif in its coiled-coil domain. Slowing the rate of internalization by co-assembly with R2 represents a novel role for GPCR heterodimerization whereby R2 subunits, via their C terminus coiled-coil domain, mask a dileucine motif on R1a subunits to determine the surface stability of the GABA(B) receptor.     

Structure of GABAB receptors in rat retina

In the search for yet unknown subtypes of GABAB receptors, the subunit architecture of GABAB receptors in the retina was analyzed using selective antisera. Immunopurification of the splice variants GABAB1a and GABAB1b demonstrated that both were associated with GABAB2. Quantitative immunoprecipitation experiments indicated that practical the entire GABAB receptor population in the retina consists of the receptor subtypes GABAB1a/GABAB2 and GABAB1b/GABAB2, although low levels of GABAB1c/GABAB2 cannot be excluded. The data rule out the existence of GABAB receptors containing the splice variants GABAB1d and GABAB1e. Moreover, no evidence for homomeric GABAB1 receptors was found. Among the splice variants, GABAB1a is by far the predominant one in neonatal and adult retina, whereas GABAB1b is expressed only late in postnatal development and in the adult retina. Since GABAB1a is expressed at high levels before functional synapses are formed, this specific receptor subtype might be involved in the maturation of the retina. Finally, subcellular fractionation demonstrated that GABAB1a, but not GABAB1b, is present in postsynaptic densities, suggesting a differential pre- and postsynaptic localisation of both splice variants.     

Impairment of GABAB receptor dimer by endogenous 14-3-3ζ in chronic pain conditions

In the central nervous system, the inhibitory GABAB receptor is the archetype of heterodimeric G protein-coupled receptors (GPCRs). However, the regulation of GABAB dimerization, and more generally of GPCR oligomerization, remains largely unknown. We propose a novel mechanism for inhibition of GPCR activity through de-dimerization in pathological conditions. We show here that 14-3-3ζ, a GABAB1-binding protein, dissociates the GABAB heterodimer, resulting in the impairment of GABAB signalling in spinal neurons. In the dorsal spinal cord of neuropathic rats, 14-3-3ζ is overexpressed and weakens GABAB inhibition. Using anti-14-3-3ζ siRNA or competing peptides disrupts 14-3-3ζ/GABAB1 interaction and restores functional GABAB heterodimers in the dorsal horn. Importantly, both strategies greatly enhance the anti-nociceptive effect of intrathecal Baclofen in neuropathic rats. Taken together, our data provide the first example of endogenous regulation of a GPCR oligomeric state and demonstrate its functional impact on the pathophysiological process of neuropathic pain sensitization.     

Dynamic interaction between soluble tubulin and C-terminal domains of N-methyl-D-aspartate receptor subunits

The cytoplasmic C-terminal domains (CTs) of the NR1 and NR2 subunits of the NMDA receptor have been implicated in its anchoring to the subsynaptic cytoskeleton. Here, we used affinity chromatography with glutathione S-transferase-NR1-CT and -NR2B-CT fusion proteins to identify novel binding partner(s) of these NMDA receptor subunits. Upon incubation with rat brain cytosolic protein fraction, both NR1-CT and NR2B-CT, but not glutathione S-transferase, specifically bound tubulin. The respective fusion proteins also bound tubulin purified from brain, suggesting a direct interaction between the two binding partners. In tubulin polymerization assays, NR1-CT and NR2B-CT significantly decreased the rate of microtubule formation without destabilizing preformed microtubules. Moreover, only minor fractions of either fusion protein coprecipitated with the newly formed microtubules. Consistent with these findings, ultrastructural analysis of the newly formed microtubules revealed a limited association only with the CTs of the NR1 and NR2B. These data suggest a direct interaction of the NMDA receptor channel subunit CTs and tubulin dimers or soluble forms of tubulin. The efficient modulation of microtubule dynamics by the NR1 and NR2 cytoplasmic domains suggests a functional interaction of the receptor and the subsynaptic cytoskeletal network that may play a role during morphological adaptations, as observed during synaptogenesis and in adult CNS plasticity.     

Intracellular coiled-coil domain engaged in subunit interaction and assembly of melastatin-related transient receptor potential channel 2

TRPM2 channels, activated by adenosine diphosphoribose and related molecules, are assembled as oligomers and most likely tetramers. However, the molecular determinants driving the subunit interaction and assembly of the TRPM2 channels are not well defined. Here we examined, using site-directed mutagenesis in conjunction with co-immunoprecipitation and patch clamp recording, the role of a coiled-coil domain in the intracellular C terminus of TRPM2 subunit in subunit interaction and channel assembly. Deletion of the coiled-coil domain resulted in severe disruption of the subunit interaction and substantial loss of the adenosine diphosphoribose-evoked channel currents. Individual or combined mutations to glutamine of the hydrophobic residues at positions a and d of the abcdef heptad repeat, key residues for protein-protein interaction, significantly reduced the subunit interaction and channel currents; the mutational effects on the subunit interaction and channel currents were clearly correlated. Furthermore, deletion of the coiled-coil domain in a pore mutant subunit abolished its dominant negative phenotypic functional suppression. These results provide strong evidence that the coiled-coil domain is critically engaged in the TRPM2 subunit interaction and such interaction is required for assembly of functional TRPM2 channel. The coiled-coil domain, which is highly conserved within the TRPM subfamily, may serve as a general structural element governing the assembly of TRPM channels.     

A site-directed cross-linking approach to the characterization of subunit E-subunit G contacts in the vacuolar H+-ATPase stator

Abstract

To operate as a rotary motor, the ATP-hydrolyzing domain of the vacuolar H⁺-ATPase must be connected to a fixed structure in its membrane-bound proton pump domain by a mechanical stator. Although low-resolution structural data and spectroscopic analysis indicate that a filament-like subunit E/subunit G heterodimer performs this role, more detailed information about the relative arrangement of these subunits is limited. We have used a site-directed cross-linking approach to show that, in both bacterial and yeast V-type ATPases, the N-terminal α-helical segments of the G and E subunits are closely aligned over a distance of up to 40 Å. Furthermore, cross-linking coupled to mass spectrometry shows that the C-terminal end of G is anchored at the C-terminal globular domain of subunit E. These data are consistent with a stator model comprising two ～ 150 Å long parallel α-helices linked to each other at both ends, stabilized by a coiled-coil arrangement and capped by the globular C-terminal domain of E that connects the cytoplasmic end of the helical structure to the V-ATPase catalytic domain.     

A normal mode analysis of structural plasticity in the biomolecular motor F(1)-ATPase

Normal modes have been used to explore the inherent flexibility of the alpha, beta and gamma subunits of F(1)-ATPase in isolation and as part of the alpha(3)beta(3)gamma complex. It was found that the structural plasticity of the gamma and beta subunits, in particular, correlates with their functions. The N and C-terminal helices forming the coiled-coil domain of the gamma subunit are highly flexible in the isolated subunit, but more rigid in the alpha(3)beta(3)gamma complex due to interactions with other subunits. The globular domain of the gamma subunit is structurally relatively rigid when isolated and in the alpha(3)beta(3)gamma complex; this is important for its functional role in coupling the F(0) and F(1) complex of ATP synthase and in inducing the conformational changes of the beta subunits in synthesis. Most important, the character of the lowest-frequency modes of the beta(E) subunit is highly correlated with the large beta(E) --> beta(TP) transition. This holds for the C-terminal domain and the nucleotide-binding domain, which undergo significant conformational transitions in the functional cycle of F(1)-ATPase. This is most evident in the ligand-free beta(E) subunit; the flexibility in the nucleotide-binding domain is reduced somewhat in the beta(TP) subunit in the presence of Mg(2+).ATP. The low-frequency modes of the alpha(3)beta(3)gamma complex show that the motions of the globular domain of the gamma subunit and of the C-terminal and nucleotide binding domains of the beta(E) subunits are coupled, in accord with their function. Overall, the normal mode analysis reveals that F(1)-ATPase, like other macromolecular assemblies, has the intrinsic structural flexibility required for its function encoded in its sequence and three-dimensional structure. This inherent plasticity is an essential aspect of assuring a small free energy cost for the large-scale conformational transition that occurs in molecular motors.     

Coiled-coil interactions modulate multimerization, mitochondrial binding and kinase activity of myotonic dystrophy protein kinase splice isoforms

The myotonic dystrophy protein kinase polypeptide repertoire in mice and humans consists of six different splice isoforms that vary in the nature of their C-terminal tails and in the presence or absence of an internal Val-Ser-Gly-Gly-Gly motif. Here, we demonstrate that myotonic dystrophy protein kinase isoforms exist in high-molecular-weight complexes controlled by homo- and heteromultimerization. This multimerization is mediated by coiled-coil interactions in the tail-proximal domain and occurs independently of alternatively spliced protein segments or myotonic dystrophy protein kinase activity. Complex formation was impaired in myotonic dystrophy protein kinase mutants in which three leucines at positions a and d in the coiled-coil heptad repeats were mutated to glycines. These coiled-coil mutants were still capable of autophosphorylation and transphosphorylation of peptides, but the rates of their kinase activities were significantly lowered. Moreover, phosphorylation of the natural myotonic dystrophy protein kinase substrate, myosin phosphatase targeting subunit, was preserved, even though binding of the myotonic dystrophy protein kinase to the myosin phosphatase targeting subunit was strongly reduced. Furthermore, the association of myotonic dystrophy protein kinase isoform C to the mitochondrial outer membrane was weakened when the coiled-coil interaction was perturbed. Our findings indicate that the coiled-coil domain modulates myotonic dystrophy protein kinase multimerization, substrate binding, kinase activity and subcellular localization characteristics.     

Requirement for the N-Terminal Coiled-Coil Domain for Expression and Function,but not Subunit Interaction of,the ADPR-Activated TRPM2 Channel

Transient receptor potential melastatin 2 (TRPM2) proteins form multiple-subunit complexes, most likely homotetramers, which operate as Ca²⁺-permeable, nonselective cation channels activated by intracellular ADP-ribose (ADPR) and oxidative stress. Each TRPM2 channel subunit is predicted to contain two coiled-coil (CC) domains, one in the N-terminus and the other in the C-terminus. Our recent study has shown that the C-terminal CC domain plays an important, but not exclusive, role in the TRPM2 channel assembly. This study aimed to examine the potential role of the N-terminal CC domain. Domain deletion dramatically reduced protein expression and abolished ADPR-evoked currents but did not alter the subunit interaction. Deletion of both CC domains strongly attenuated the subunit interaction, confirming that the C-terminal CC domain is critical in the subunit interaction. Glutamine substitutions into individual hydrophobic residues at positions a and d in the heptad repeats to disrupt the CC formation had no effect on protein expression, subunit interaction, or ADPR-evoked currents. Mutation of Ile⁶⁵⁸ to glutamine, which did not perturb the CC formation, decreased ADPR-evoked currents without affecting protein expression, subunit interaction, or membrane trafficking. These results collectively suggest the requirement for the N-terminal CC domain for protein expression and function, but not subunit interaction, of the TRPM2 channel.     

GABAB受体研究现状

代谢型GABAB受体是C族蛋白偶联受体中的特殊的异源二聚体成员,由GABAB1亚基和GABAB2亚基组成。每个亚基在受体的激活过程中具有各自不同的功效。初步研究发现GABAB受体的活性下调方式如失敏和内化并不遵从经典的路径。近期的研究还发现GABAB受体新的生理功能,GABAB1亚基的不同亚型体在认知学习中具有重要的作用。