Is Ca2+ -Calmodulin-Dependent Protein Phosphorylation in Rat Brain Modulated by Carboxylmethylation?

Calmodulin stimulation of protein kinase activity in calmodulin-depleted preparations of rat brain cytosol or synaptosomal membranes was attenuated by prior carboxylmethylation of the enzyme source with purified protein-O-carboxylmethyltransferase. Similarly, calmodulin stimulation of highly purified Ca2+-calmodulin-dependent protein kinase was reduced if the kinase was exposed to methylating conditions prior to addition of calmodulin. Biochemical and acidic sodium dodecyl sulfate-gel electrophoretic analyses indicated that all sources of protein kinase activity were substrates for methylation. The specific activity of methyl group incorporation into protein kinase increased with increasing purity of the preparation, reaching values of 1.72 pmol CH3/micrograms protein or 0.15-1.12 mol CH3/mol of holoenzyme. Analysis of ATP binding in cytosol with the use of the photoaffinity probe [32P]8-azido-ATP indicated that carboxylmethylation reduced ATP binding. These results suggest that carboxylmethylation of Ca2+-calmodulin protein kinase may modulate the activity of this enzyme in rat brain.     

Evidence for β-adrenergic activation of Na+-dependent efflux of Ca2+ from isolated liver mitochondria

The existence of a Na⁺-dependent mechanism for Ca²⁺ efflux from isolated rat liver mitochondria was confirmed. The activity of this system is decreased by 60% in mitochondria isolated from perfused livers. The Na⁺-dependent activity is fully restored by infusion of either 1μm-adrenaline or 1μm-isoprenaline, but the α-adrenergic agonist phenylephrine is ineffective.     

Calretinin Regulates Ca2+-dependent Inactivation and Facilitation of Cav2.1 Ca2+ Channels through a Direct Interaction with the α12.1 Subunit

Voltage-gated Ca_v2.1 Ca²⁺ channels undergo dual modulation by Ca²⁺, Ca²⁺-dependent inactivation (CDI), and Ca²⁺-dependent facilitation (CDF), which can influence synaptic plasticity in the nervous system. Although the molecular determinants controlling CDI and CDF have been the focus of intense research, little is known about the factors regulating these processes in neurons. Here, we show that calretinin (CR), a Ca²⁺-binding protein highly expressed in subpopulations of neurons in the brain, inhibits CDI and enhances CDF by binding directly to α₁2.1. Screening of a phage display library with CR as bait revealed a highly basic CR-binding domain (CRB) present in multiple copies in the cytoplasmic linker between domains II and III of α₁2.1. In pulldown assays, CR binding to fusion proteins containing these CRBs was largely Ca²⁺-dependent. α₁2.1 coimmunoprecipitated with CR antibodies from transfected cells and mouse cerebellum, which confirmed the existence of CR-Ca_v2.1 complexes in vitro and in vivo. In HEK293T cells, CR significantly decreased Ca_v2.1 CDI and increased CDF. CR binding to α₁2.1 was required for these effects, because they were not observed upon substitution of the II-III linker of α₁2.1 with that from the Ca_v1.2 α₁ subunit (α₁1.2), which lacks the CRBs. In addition, coexpression of a protein containing the CRBs blocked the modulatory action of CR, most likely by competing with CR for interactions with α₁2.1. Our findings highlight an unexpected role for CR in directly modulating effectors such as Ca_v2.1, which may have major consequences for Ca²⁺ signaling and neuronal excitability.     

Nol9 Is a Spatial Regulator for the Human ITS2 Pre-rRNA Endonuclease–Kinase Complex

The ribosome plays a universal role in translating the cellular proteome. Defects in the ribosome assembly factor Las1L are associated with congenital lethal motor neuron disease and X-linked intellectual disability disorders, yet its role in processing precursor ribosomal RNA (pre-rRNA) is largely unclear. The Las1L endoribonuclease associates with the Nol9 polynucleotide kinase to form the internal transcribed spacer 2 (ITS2) pre-rRNA endonuclease–kinase machinery. Together, Las1L–Nol9 catalyzes RNA cleavage and phosphorylation to mark the ITS2 for degradation. While ITS2 processing is critical for the production of functional ribosomes, the regulation of mammalian Las1L–Nol9 remains obscure. Here we characterize the human Las1L–Nol9 complex and identify critical molecular features that regulate its assembly and spatial organization. We establish that Las1L and Nol9 form a higher-order complex and identify the regions responsible for orchestrating this intricate architecture. Structural analysis by high-resolution imaging defines the intricate spatial pattern of Las1L–Nol9 within the nucleolar sub-structure linked with late pre-rRNA processing events. Furthermore, we uncover a Nol9-encoded nucleolar localization sequence that is responsible for nucleolar transport of the assembled Las1L–Nol9 complex. Together, these data provide a mechanism for the assembly and nucleolar localization of the human ITS2 pre-rRNA endonuclease–kinase complex.     

Adaptor Protein Complex 2–Mediated Endocytosis Is Crucial for Male Reproductive Organ Development in Arabidopsis

Fertilization in flowering plants requires the temporal and spatial coordination of many developmental processes, including pollen production, anther dehiscence, ovule production, and pollen tube elongation. However, it remains elusive as to how this coordination occurs during reproduction. Here, we present evidence that endocytosis, involving heterotetrameric adaptor protein complex 2 (AP-2), plays a crucial role in fertilization. An Arabidopsis thaliana mutant ap2m displays multiple defects in pollen production and viability, as well as elongation of staminal filaments and pollen tubes, all of which are pivotal processes needed for fertilization. Of these abnormalities, the defects in elongation of staminal filaments and pollen tubes were partially rescued by exogenous auxin. Moreover, DR5rev:GFP (for green fluorescent protein) expression was greatly reduced in filaments and anthers in ap2m mutant plants. At the cellular level, ap2m mutants displayed defects in both endocytosis of N-(3-triethylammonium-propyl)-4-(4-diethylaminophenylhexatrienyl) pyridinium dibromide, a lypophilic dye used as an endocytosis marker, and polar localization of auxin-efflux carrier PIN FORMED2 (PIN2) in the stamen filaments. Moreover, these defects were phenocopied by treatment with Tyrphostin A23, an inhibitor of endocytosis. Based on these results, we propose that AP-2–dependent endocytosis plays a crucial role in coordinating the multiple developmental aspects of male reproductive organs by modulating cellular auxin level through the regulation of the amount and polarity of PINs.     

A Class VI Unconventional Myosin Is Associated with a Homologue of a Microtubule-binding Protein,Cytoplasmic Linker Protein–170, in Neurons and at the Posterior Pole of Drosophila Embryos

Abstract. Coordination of cellular organization requires the interaction of the cytoskeletal filament systems. Recently, several lines of investigation have suggested that transport of cellular components along both microtubules and actin filaments is important for cellular organization and function. We report here on molecules that may mediate coordination between the actin and microtubule cytoskeletons. We have identified a 195-kD protein that coimmunoprecipitates with a class VI myosin, Drosophila 95F unconventional myosin. Cloning and sequencing of the gene encoding the 195-kD protein reveals that it is the first homologue identified of cytoplasmic linker protein (CLIP)–170, a protein that links endocytic vesicles to microtubules. We have named this protein D-CLIP-190 (the predicted molecular mass is 189 kD) based on its similarity to CLIP-170 and its ability to cosediment with microtubules. The similarity between D-CLIP-190 and CLIP-170 extends throughout the length of the proteins, and they have a number of predicted sequence and structural features in common. 95F myosin and D-CLIP-190 are coexpressed in a number of tissues during embryogenesis in Drosophila. In the axonal processes of neurons, they are colocalized in the same particulate structures, which resemble vesicles. They are also colocalized at the posterior pole of the early embryo, and this localization is dependent on the actin cytoskeleton. The association of a myosin and a homologue of a microtubule-binding protein in the nervous system and at the posterior pole, where both microtubule and actin-dependent processes are known to be important, leads us to speculate that these two proteins may functionally link the actin and microtubule cytoskeletons.Global organization of the cell and the coordination of its physiology requires interaction between different cytoskeletal systems. During interphase, a typical eukaryotic cell has microtubules emanating from the centrosome located near the nucleus, which extend to the periphery of the cell, presumably interacting with the cortical actin filament meshwork. Microtubules during interphase are thought to be mainly required for the organization of the membrane systems (e.g., vesicular traffic and organelle movement). The actin-rich cortex is important for maintaining cell shape and for cellular movement.There is increasing evidence of coordination between the actin and the microtubule cytoskeletons (Langford, 1995; Koonce, 1996). Data from a number of systems suggests that many cell types use a combination of microtubule and actin filament–based transport in vesicle and organelle trafficking. It is well established that microtubules are required for long distance transport of cellular components. In contrast, the actin cytoskeleton is thought to be required for more local traffic. The best evidence for transport along both cytoskeletal systems is in neurons. Vesicles appear to be transported along actin filaments in mammalian growth cones (Evans and Bridgman, 1995). Furthermore, gelsolin, which promotes depolymerization of actin filaments, has been shown to inhibit fast axonal transport in this system (Brady et al., 1984). In extruded squid axoplasm, Kuznetsov et al. (1992) observed what appeared to be the same vesicle moving along microtubules and then, subsequently, along microfilaments. Inhibitor studies provide evidence that mitochondria can move along both actin filaments and microtubules in neurons in vivo (Morris and Hollenbeck, 1995). These data support the idea that actin filament and microtubule-based transport cooperate to achieve proper organization of cellular components.The same phenomenon may be occurring in other cell types. In yeast, the mutant phenotype of the MYO2 gene, which encodes an unconventional myosin, is suppressed by overexpression of a kinesin-related protein. These two proteins are colocalized in regions of active growth where a polarized arrangement of actin plays an important role (Lillie and Brown, 1992, 1994). Microtubules are not normally required for this growth. Thus, the basis for suppression is not completely understood. However, the phenotypic suppression suggests that perhaps microtubule-based transport can substitute for actin filament–based transport, under some conditions. In polarized epithelial cells, Fath et al. (1994) have isolated a population of vesicles containing both myosin and microtubule motors. They speculate that proper transport of vesicles relies on both microtubule and actin filament–based transport.Previously, it has been shown that a class VI unconventional myosin, the Drosophila 95F unconventional myosin, transports particles along actin filaments during the syncytial blastoderm stage of Drosophila embryonic development (Mermall et al., 1994). 95F myosin activity is required for normal embryonic development (Mermall and Miller, 1995). 95F myosin is also associated with particulate structures in other cells of the embryo later in development where it may also be involved in actin-based transport. To investigate further the transport catalyzed by 95F myosin, we have begun studies to identify proteins associated with 95F myosin that might be cargoes or regulators. In this work, we have identified a protein that coimmunoprecipitates with 95F myosin. Sequence analysis reveals that this protein is the Drosophila homologue of cytoplasmic linker protein (CLIP)¹–170. CLIP-170 is believed to function as a linker between endocytic vesicles and microtubules (Pierre et al., 1992). We have named this associated protein D-CLIP-190. Colocalization of 95F myosin and D-CLIP-190 at the subcellular level at several times in development and in cultured embryonic cells provides support for the in vivo association of these two proteins. The association of a myosin and a homologue of a microtubule-binding protein suggests that these two proteins may act to coordinate the interaction between actin filaments and microtubules.     

Reconstitution of the Peridinin–chlorophyll a Protein (PCP): Evidence for Functional Flexibility in Chlorophyll Binding

The coding regions for the N-domain, and full length peridinin–chlorophyll a apoprotein (full length PCP), were expressed in Escherichia coli. The apoproteins formed inclusion bodies from which the peptides could be released by hot buffer. Both the above constructs were reconstituted by addition of a total pigment extract from native PCP. After purification by ion exchange chromatography, the absorbance, fluorescence excitation and CD spectra resembled those of the native PCP. Energy transfer from peridinin to Chl a was restored and a specific fluorescence activity calculated which was ~86% of that of native PCP. Size exclusion analysis and CD spectra showed that the N-domain PCP dimerized on reconstitution. Chl a could be replaced by Chl b, 3-acetyl Chl a, Chl d and Bchl using the N-domain apo protein. The specific fluorescence activity was the same for constructs with Chl a, 3-acetyl Chl a, and Chl d but significantly reduced for those made with Chl b. Reconstitutions with mixtures of chlorophylls were also made with eg Chl b and Chl d and energy transfer from the higher energy Qy band to the lower was demonstrated.     

Rotation of the γ Subunit in F1-ATPase; Evidence That ATP Synthase Is a Rotary Motor Enzyme

ATP-dependent, azide-sensitive rotation of the subunit relative to the ₃₃ hexagonal ring of ATP synthase was observed with a single molecule imaging system. Thus, ATP synthase is a rotary motor enzyme, the first ever found.     

Is the Ca2+-ATPase from sarcoplasmic reticulum also a heat pump?

We calculate, using the first law of thermodynamics, the membrane heat fluxes during active transport of Ca²⁺ in the Ca²⁺-ATPase in leaky and intact vesicles, during ATP hydrolysis or synthesis conditions. The results show that the vesicle interior may cool down during hydrolysis and Ca²⁺-uptake, and heat up during ATP synthesis and Ca²⁺-efflux. The heat flux varies with the SERCA isoform. Electroneutral processes and rapid equilibration of water were assumed. The results are consistent with the second law of thermodynamics for the overall processes. The expression for the heat flux and experimental data, show that important contributions come from the enthalpy of hydrolysis for the medium in question, and from proton transport between the vesicle interior and exterior. The analysis give quantitative support to earlier proposals that certain, but not all, Ca²⁺-ATPases, not only act as Ca²⁺-pumps, but also as heat pumps. It can thus help explain why SERCA 1 type enzymes dominate in tissues where thermal regulation is important, while SERCA 2 type enzymes, with their lower activity and better ability to use the energy from the reaction to pump ions, dominate in tissues where this is not an issue.

Ca2+-dependent Inactivation of CaV1.2 Channels Prevents Gd3+ Block: Does Ca2+ Block the Pore of Inactivated Channels?

Lanthanide gadolinium (Gd(3+)) blocks Ca(V)1.2 channels at the selectivity filter. Here we investigated whether Gd(3+) block interferes with Ca(2+)-dependent inactivation, which requires Ca(2+) entry through the same site. Using brief pulses to 200 mV that relieve Gd(3+) block but not inactivation, we monitored how the proportions of open and open-blocked channels change during inactivation. We found that blocked channels inactivate much less. This is expected for Gd(3+) block of the Ca(2+) influx that enhances inactivation. However, we also found that the extent of Gd(3+) block did not change when inactivation was reduced by abolition of Ca(2+)/calmodulin interaction, showing that Gd(3+) does not block the inactivated channel. Thus, Gd(3+) block and inactivation are mutually exclusive, suggesting action at a common site. These observations suggest that inactivation causes a change at the selectivity filter that either hides the Gd(3+) site or reduces its affinity, or that Ca(2+) occupies the binding site at the selectivity filter in inactivated channels. The latter possibility is supported by previous findings that the EEQE mutation of the selectivity EEEE locus is void of Ca(2+)-dependent inactivation (Zong Z.Q., J.Y. Zhou, and T. Tanabe. 1994. Biochem. Biophys. Res. Commun. 201:1117-11123), and that Ca(2+)-inactivated channels conduct Na(+) when Ca(2+) is removed from the extracellular medium (Babich O., D. Isaev, and R. Shirokov. 2005. J. Physiol. 565:709-717). Based on these results, we propose that inactivation increases affinity of the selectivity filter for Ca(2+) so that Ca(2+) ion blocks the pore. A minimal model, in which the inactivation "gate" is an increase in affinity of the selectivity filter for permeating ions, successfully simulates the characteristic U-shaped voltage dependence of inactivation in Ca(2+).     

Is a Ca2+ -ATPase involved in Ca2+ regulation during capacitation and the acrosome reaction of guinea-pig spermatozoa?

The Ca2+-ATPase antagonists quercetin and ethacrynic acid accelerated the onset of the acrosome reaction in guinea-pig spermatozoa incubated in the continuous presence of Ca2+, whereas furosemide had no effect, and sodium orthovanadate only affected sperm motility. When spermatozoa were preincubated in a 'Ca2+-free' medium, quercetin and ethacrynic acid shortened capacitation time: spermatozoa incubated for 1 h in 100-200 microM-ethacrynic acid showed 60-80% acrosome reactions when Ca2+ was added. Such spermatozoa were able to fertilize zona-free hamster eggs. Our results therefore point to the possible involvement of a Ca2+-ATPase in the regulation of intracellular Ca2+ in spermatozoa. Cysteine and dithiothreitol, both disulphide reducing agents, prevented the effects of quercetin and ethacrynic acid, suggesting that sulphydryl groups may be important for the expression of Ca2+-ATPase activity. Lysophosphatidylserine (LS) also prevented the stimulatory effect of ethacrynic acid, an effect similar to that shown by LS on lysophosphatidylcholine (LC). It is argued that both LS and LC could exert their action through an effect on the Ca2+-ATPase.     

Production of a Monoclonal Antibody Targeting the M Protein of MERS-CoV for Detection of MERS-CoV Using a Synthetic Peptide Epitope Formulated with a CpG–DNA–Liposome Complex

The Middle East respiratory syndrome-related coronavirus (MERS-CoV) contains four major structural proteins, the spike glycoprotein, nucleocapsid phosphoprotein, membrane (M) glycoprotein and small envelope glycoprotein. The M protein of MERS-CoV has a role in the morphogenesis or assembly of the virus and inhibits type I interferon expression in infected cells. Here, we produced a monoclonal antibody specific against the M protein of MERS-CoV by injecting BALB/c mice with a complex containing the epitope peptide and CpG–DNA encapsulated with a phosphatidyl-β-oleoyl-γ-palmitoyl ethanolamine (DOPE):cholesterol hemisuccinate (CHEMS). The monoclonal antibody was reactive to the epitope peptide of the M protein of MERS-CoV which was confirmed by western blotting and immunoprecipitations. Indirect immunofluorescence assay and confocal image analysis showed that the monoclonal antibody binds specifically to the M protein of MERS-CoV in the virus-infected cells. Further studies using this monoclonal antibody may provide important information on the function of the M protein and its future application in diagnostics.

     

The Ca<Superscript>2+</Superscript>-dependent Activator Protein for Secretion CAPS: Do I Dock or do I Prime?

The “Ca²⁺-dependent activator protein for secretion” (CAPS) is a protein which reconstitutes regulated secretion in permeabilized neuroendocrine cells. It is generally accepted that CAPS plays an important role in the release of the contents of dense core vesicles in the nervous system as well as in a variety of other secretory tissues. At which step in the exocytotic process CAPS functions as well as its role in the fusion of synaptic vesicles is still under dispute. A recent growth spurt in the CAPS field has been fueled by genetic approaches in Caenorhabditis elegans and Drosophila as well as the application of knockout and knockdown approaches in mouse cells and in cell lines, respectively. We have attempted to review the body of work that established CAPS as an important regulator of secretion and to describe new information that has furthered our understanding of how CAPS may function. We discuss the conclusions, point out areas where controversy remains, and suggest directions for future experiments.     

Is intracellular Ca2+ the trigger for oxygen radical production by polymorphonuclear leucocytes?

The aim of this paper is critically to evaluate the existing evidence for the role of intracellular Ca2+ in polymorphonuclear leucocyte (PMN) activation and in particular in oxygen radical production. Indirect experiments are based on the manipulation of extracellular Ca2+, measurement of 45Ca fluxes, employing pharmacological agents such as Ca2+-ionophores and intracellular Ca2+ antagonists and monitoring chlortetracycline fluorescence. Experiments of this type do not provide the necessary definitive evidence that an increase in intracellular Ca2+ is the trigger for PMN activation. Recent direct measurements of intracellular free Ca2+ using the Ca2+-activated photoprotein, obelin, and the Ca2+-sensitive fluorescent indicator, quin 2, have provided evidence for the existence of two distinct mechanisms of activation, one triggered by a rise in intracellular Ca2+ and the other independent of a rise in intracellular Ca2+. The source of the Ca2+ for the former mechanism is mainly extracellular but can also come from an intracellular Ca2+ store.     

Quantitative Proteomics Reveals Protein–Protein Interactions with Fibroblast Growth Factor 12 as a Component of the Voltage-Gated Sodium Channel 1.2 (Nav1.2) Macromolecular Complex in Mammalian Brain

Voltage-gated sodium channels (Nav1.1–Nav1.9) are responsible for the initiation and propagation of action potentials in neurons, controlling firing patterns, synaptic transmission and plasticity of the brain circuit. Yet, it is the protein–protein interactions of the macromolecular complex that exert diverse modulatory actions on the channel, dictating its ultimate functional outcome. Despite the fundamental role of Nav channels in the brain, information on its proteome is still lacking. Here we used affinity purification from crude membrane extracts of whole brain followed by quantitative high-resolution mass spectrometry to resolve the identity of Nav1.2 protein interactors. Of the identified putative protein interactors, fibroblast growth factor 12 (FGF12), a member of the nonsecreted intracellular FGF family, exhibited 30-fold enrichment in Nav1.2 purifications compared with other identified proteins. Using confocal microscopy, we visualized native FGF12 in the brain tissue and confirmed that FGF12 forms a complex with Nav1.2 channels at the axonal initial segment, the subcellular specialized domain of neurons required for action potential initiation. Co-immunoprecipitation studies in a heterologous expression system validate Nav1.2 and FGF12 as interactors, whereas patch-clamp electrophysiology reveals that FGF12 acts synergistically with CaMKII, a known kinase regulator of Nav channels, to modulate Nav1.2-encoded currents. In the presence of CaMKII inhibitors we found that FGF12 produces a bidirectional shift in the voltage-dependence of activation (more depolarized) and the steady-state inactivation (more hyperpolarized) of Nav1.2, increasing the channel availability. Although providing the first characterization of the Nav1.2 CNS proteome, we identify FGF12 as a new functionally relevant interactor. Our studies will provide invaluable information to parse out the molecular determinant underlying neuronal excitability and plasticity, and extending the relevance of iFGFs signaling in the normal and diseased brain.Voltage-gated sodium channels (Nav)¹ are transmembrane proteins consisting of a pore-forming α subunit (Nav1.1-Nav1.9) and one or more accessory β-subunits (β₁–β₄) (1–3). Predominately clustered at the axonal initial segment (AIS), the α subunit alone is necessary and sufficient for channel assembly and the initiation and propagation of action potentials following membrane depolarization (4). Although the α subunit is functional on its own, it is the transient and stable protein–protein interactions that modulate subcellular trafficking, compartmentalization, functional expression, and fine-tune the channel biophysical properties (5–9). Thus, the Nav channel and the protein constituents that comprise the protein–protein interaction network are all part of a macromolecular complex that modulates the spatiotemporal dynamics of neuronal input and output playing a critical role in synaptic transmission, signal integration, and neuronal plasticity. Perturbations in this protein–protein interaction network can lead to deficits in neuronal excitability, and eventually neurodegeneration and cell death (10–15).Given the relevance of these interactions for the native channel activity and its overall role in controlling brain circuits, it is increasingly important to uncover these associations. Antibody-based affinity purification (AP) combined with mass spectrometry (MS) is widely used for the enrichment and analysis of target proteins and constituents of their protein–protein interactions as it can be performed at near physiological conditions and preserves post-translational modifications relevant to protein complex organization (16–19). Differential mass spectrometry provides an unbiased method for the efficient, MS-based measurement of relative protein fold changes across multiple complex biological samples. This technology has been successfully applied to a number of ion channels (20–26), but—to the best of our knowledge—not to the study of any member of the Nav channel family. Using a target-directed AP approach combined with quantitative MS, we identified proteins constituting the putative interactome of Nav1.2, one of three dominant Nav channel isoforms in the mammalian brain, from native tissue (1, 2, 4, 8). Among these putative interactors, the fibroblast growth factor 12 (FGF12), a member of the intracellular FGF family (5, 13, 14), stood out as one of the most abundant coprecipitating proteins with ∼30-fold enrichment over other interactors. With a combination of confocal microscopy in brain tissue, reconstitution of the interactor in a heterologous systems and electrophysiological assays, we provide validation for FGF12 as a bona fide relevant component of the Nav1.2 proteome and a modulator of Nav1.2-encoded currents. Altogether, the identified channel/protein interaction between FGF12 and Nav1.2 provides new insights for structural and functional interpretation of neuronal excitability, synaptic transmission, and plasticity in the normal and diseased brain.     

Ric-8A,a GEF,and a Chaperone for G Protein α-Subunits: Evidence for the Two-Faced Interface

Resistance to inhibitors of cholinesterase 8A (Ric-8A) is a prominent non-receptor GEF and a chaperone of G protein α-subunits (Gα). Recent studies shed light on the structure of Ric-8A, providing insights into the mechanisms underlying its interaction with Gα. Ric-8A is composed of a core armadillo-like domain and a flexible C-terminal tail. Interaction of a conserved concave surface of its core domain with the Gα C-terminus appears to mediate formation of the initial Ric-8A/GαGDP intermediate, followed by the formation of a stable nucleotide-free complex. The latter event involves a large-scale dislocation of the Gα α5-helix that produces an extensive primary interface and disrupts the nucleotide-binding site of Gα. The distal portion of the C-terminal tail of Ric-8A forms a smaller secondary interface, which ostensibly binds the switch II region of Gα, facilitating binding of GTP. The two-site Gα interface of Ric-8A is distinct from that of GPCRs, and might have evolved to support the chaperone function of Ric-8A.