De Novo Mutations in GNAO1, Encoding a Gαo Subunit of Heterotrimeric G Proteins,Cause Epileptic Encephalopathy

Heterotrimeric G proteins, composed of α, β, and γ subunits, can transduce a variety of signals from seven-transmembrane-type receptors to intracellular effectors. By whole-exome sequencing and subsequent mutation screening, we identified de novo heterozygous mutations in GNAO1, which encodes a Gα_o subunit of heterotrimeric G proteins, in four individuals with epileptic encephalopathy. Two of the affected individuals also showed involuntary movements. Somatic mosaicism (approximately 35% to 50% of cells, distributed across multiple cell types, harbored the mutation) was shown in one individual. By mapping the mutation onto three-dimensional models of the Gα subunit in three different complexed states, we found that the three mutants (c.521A>G [p.Asp174Gly], c.836T>A [p.Ile279Asn], and c.572_592del [p.Thr191_Phe197del]) are predicted to destabilize the Gα subunit fold. A fourth mutant (c.607G>A), in which the Gly203 residue located within the highly conserved switch II region is substituted to Arg, is predicted to impair GTP binding and/or activation of downstream effectors, although the p.Gly203Arg substitution might not interfere with Gα binding to G-protein-coupled receptors. Transient-expression experiments suggested that localization to the plasma membrane was variably impaired in the three putatively destabilized mutants. Electrophysiological analysis showed that Gα_o-mediated inhibition of calcium currents by norepinephrine tended to be lower in three of the four Gα_o mutants. These data suggest that aberrant Gα_o signaling can cause multiple neurodevelopmental phenotypes, including epileptic encephalopathy and involuntary movements.     

Homozygous and Compound-Heterozygous Mutations in TGDS Cause Catel-Manzke Syndrome

Catel-Manzke syndrome is characterized by Pierre Robin sequence and a unique form of bilateral hyperphalangy causing a clinodactyly of the index finger. We describe the identification of homozygous and compound heterozygous mutations in TGDS in seven unrelated individuals with typical Catel-Manzke syndrome by exome sequencing. Six different TGDS mutations were detected: c.892A>G (p.Asn298Asp), c.270_271del (p.Lys91Asnfs^∗22), c.298G>T (p.Ala100Ser), c.294T>G (p.Phe98Leu), c.269A>G (p.Glu90Gly), and c.700T>C (p.Tyr234His), all predicted to be disease causing. By using haplotype reconstruction we showed that the mutation c.298G>T is probably a founder mutation. Due to the spectrum of the amino acid changes, we suggest that loss of function in TGDS is the underlying mechanism of Catel-Manzke syndrome. TGDS (dTDP-D-glucose 4,6-dehydrogenase) is a conserved protein belonging to the SDR family and probably plays a role in nucleotide sugar metabolism.     

Somatic Activating PIK3CA Mutations Cause Venous Malformation

Somatic mutations in TEK, the gene encoding endothelial cell tyrosine kinase receptor TIE2, cause more than half of sporadically occurring unifocal venous malformations (VMs). Here, we report that somatic mutations in PIK3CA, the gene encoding the catalytic p110α subunit of PI3K, cause 54% (27 out of 50) of VMs with no detected TEK mutation. The hotspot mutations c.1624G>A, c.1633G>A, and c.3140A>G (p.Glu542Lys, p.Glu545Lys, and p.His1047Arg), frequent in PIK3CA-associated cancers, overgrowth syndromes, and lymphatic malformation (LM), account for >92% of individuals who carry mutations. Like VM-causative mutations in TEK, the PIK3CA mutations cause chronic activation of AKT, dysregulation of certain important angiogenic factors, and abnormal endothelial cell morphology when expressed in human umbilical vein endothelial cells (HUVECs). The p110α-specific inhibitor BYL719 restores all abnormal phenotypes tested, in PIK3CA- as well as TEK-mutant HUVECs, demonstrating that they operate via the same pathogenic pathways. Nevertheless, significant genotype-phenotype correlations in lesion localization and histology are observed between individuals with mutations in PIK3CA versus TEK, pointing to gene-specific effects.     

Could a defective epithelial sodium channel lead to bronchiectasis

Background

Bronchiectasis is defined as a permanent dilation of the airways arising from chronic bronchial inflammation/infection. In 50% of cases, no etiology can be identified. Recently, the role of the epithelial sodium channel ENaC has been pointed out in the pathophysiology of cystic fibrosis, a disease due to mutations in the CFTR gene and causing bronchiectasis in the airways. Moreover, it was found that transgenic mice overexpressing ENaCβ present cystic fibrosis-like lung disease symptoms. Our aim was to evaluate if a defective ENaC protein could be involved in the development of bronchiectasis.

Methods

We extensively analysed ENaCβ and γ genes in 55 patients with idiopathic bronchiectasis and without two mutations in the coding regions of CFTR. Thirty-eight patients presented functional abnormalities suggesting impaired sodium transport (abnormal sweat chloride concentration or nasal potential difference measurement), and 17 had no such evidence.

Results

Sequencing of the exons and flanking introns of the ENaCβ and γ gene identified five different amino-acid changes (p.Ser82Cys, p.Pro369Thr, p.Asn288Ser in ENaCβ ; and p.Gly183Ser, p.Glu197Lys in ENaCγ) in heterozygous state in 8 patients. The p.Ser82Cys amino-acid change was found in 3 unrelated patients who were also heterozygous for a CFTR mutation or variant (1 p.F508del, 1 IVS8-5T, and 1 IVS8-5T:1716G>A (p.E528E)). The other mutations were found in patients without CFTR mutation, the p.Glu197Lys mutation in 2 patients and the other variants in single patients. Among the 8 patients bearing an ENaC mutation, 5 had functional abnormalities suggesting impaired sodium transport.

Conclusion

Our results suggest that several variants in ENaCβ and γ genes might be deleterious for ENaC function and lead to bronchiectasis, especially in patients who are trans-heterozygotes for ENaCβ/CFTR mutations or variants.     

Amino acid sequence of seminalplasmin, an antimicrobial protein from bull semen

Analytical ultracentrifugation of highly purified seminalplasmin revealed a molecular mass of 6300. Amino acid analysis of the protein preparation indicated the absence of sulfur-containing amino acids cysteine and methionine. The amino acid sequence of seminalplasmin was determined by manual Edman degradation of peptides obtained by proteolytic enzymes trypsin, chymotrypsin and thermolysin: NH₂-Ser Asp Glu Lys Ala Ser Pro Asp Lys His His Arg Phe Ser Leu Ser Arg Tyr Ala Lys Leu Ala Asn Arg Leu Ser Lys Trp Ile Gly Asn Arg Gly Asn Arg Leu Ala Asn Pro Lys Leu Leu Glu Thr Phe Lys Ser Val-COOH. The number of amino acids according to the sequence were 48, the molecular mass 6385. As predicted from the sequence, seminalplasmin very likely contains two α-helical domains in which residues 8-17 and 40-48 are involved. No evidence for the existence of β-sheet structures was obtained. Treatment of seminalplasmin with the above proteases as well as with amino peptidase M and carboxypeptidase Y completely eliminated biological activity.     

Novel three missense mutations observed in Von Hippel-Lindau gene in a patient reported with renal cell carcinoma

Von Hippel-Lindau (VHL) disease is an autosomal dominant hereditary cancer syndrome that predisposes to the development of a variety of benign and malignant tumors, especially cerebellar hemangioblastomas, retinal angiomas and clear-cell renal cell carcinomas (RCC). We have identified of VHL gene using immunohistochemistry in a patient who was diagnosed for RCC. In order to understand the involvement of mutation in the VHL gene exon 1 was amplified and sequenced (accession number: JX 401534). The sequence analysis revealed the presence of novel missense mutations c.194 C>T, c.239 G>A, c.278 G>A, c.319 C>G, c. 337 C > G leading to the following variations p.Ala 65 Val, p.Gly 80 Asp, p.Gly 93 Glu, p.Gln 107 Glu, p.Gln 113 Glu in the protein.     

Association between Rare Variants in AP4E1, a Component of Intracellular Trafficking,and Persistent Stuttering

Stuttering is a common, highly heritable neurodevelopmental disorder characterized by deficits in the volitional control of speech. Whole-exome sequencing identified two heterozygous AP4E1 coding variants, c.1549G>A (p.Val517Ile) and c.2401G>A (p.Glu801Lys), that co-segregate with persistent developmental stuttering in a large Cameroonian family, and we observed the same two variants in unrelated Cameroonians with persistent stuttering. We found 23 other rare variants, including predicted loss-of-function variants, in AP4E1 in unrelated stuttering individuals in Cameroon, Pakistan, and North America. The rate of rare variants in AP4E1 was significantly higher in unrelated Pakistani and Cameroonian stuttering individuals than in population-matched control individuals, and coding variants in this gene are exceptionally rare in the general sub-Saharan West African, South Asian, and North American populations. Clinical examination of the Cameroonian family members failed to identify any symptoms previously reported in rare individuals carrying homozygous loss-of-function mutations in this gene. AP4E1 encodes the ε subunit of the heterotetrameric (ε-β4-μ4-σ4) AP-4 complex, involved in protein sorting at the trans-Golgi network. We found that the μ4 subunit of AP-4 interacts with NAGPA, an enzyme involved in the synthesis of the mannose 6-phosphate signal that targets acid hydrolases to the lysosome and the product of a gene previously associated with stuttering. These findings implicate deficits in intracellular trafficking in persistent stuttering.     

Functional significance of conserved residues in the phosphohydrolase module of Escherichia coli MutT protein

Escherichia coli MutT protein hydrolyzes 8-oxo-7,8-dihydro-2′-dGTP (8-oxo-dGTP) to the monophosphate, thus avoiding the incorporation of 8-oxo-7,8-dihydroguanine (8-oxo-G) into nascent DNA. Bacterial and mammalian homologs of MutT protein share the phosphohydrolase module (MutT: Gly37→Gly59). By saturation mutagenesis of conserved residues in the MutT module, four of the 10 conserved residues (Gly37, Gly38, Glu53 and Glu57) were revealed to be essential to suppress spontaneous A:T→C:G transversion mutation in a mutT^– mutator strain. For the other six residues (Lys39, Glu44, Thr45, Arg52, Glu56 and Gly59), many positive mutants which can suppress the spontaneous mutation were obtained; however, all of the positive mutants for Glu44 and Arg52 either partially or inefficiently suppressed the mutation, indicating that these two residues are also important for MutT function. Several positive mutants for Lys39, Thr45, Glu56 and Gly59 efficiently decreased the elevated spontaneous mutation rate, as seen with the wild-type, hence, these four residues are non-essential for MutT function. As Lys38 and Glu55 in human MTH1, corresponding to the non-essential residues Lys39 and Glu56 in MutT, could not be replaced by any other residue without loss of function, different structural features between the two modules of MTH1 and MutT proteins are evident.     

Defective Initiation of Glycosaminoglycan Synthesis due to B3GALT6 Mutations Causes a Pleiotropic Ehlers-Danlos-Syndrome-like Connective Tissue Disorder

Proteoglycans are important components of cell plasma membranes and extracellular matrices of connective tissues. They consist of glycosaminoglycan chains attached to a core protein via a tetrasaccharide linkage, whereby the addition of the third residue is catalyzed by galactosyltransferase II (β3GalT6), encoded by B3GALT6. Homozygosity mapping and candidate gene sequence analysis in three independent families, presenting a severe autosomal-recessive connective tissue disorder characterized by skin fragility, delayed wound healing, joint hyperlaxity and contractures, muscle hypotonia, intellectual disability, and a spondyloepimetaphyseal dysplasia with bone fragility and severe kyphoscoliosis, identified biallelic B3GALT6 mutations, including homozygous missense mutations in family 1 (c.619G>C [p.Asp207His]) and family 3 (c.649G>A [p.Gly217Ser]) and compound heterozygous mutations in family 2 (c.323_344del [p.Ala108Glyfs^∗163], c.619G>C [p.Asp207His]). The phenotype overlaps with several recessive Ehlers-Danlos variants and spondyloepimetaphyseal dysplasia with joint hyperlaxity. Affected individuals’ fibroblasts exhibited a large decrease in ability to prime glycosaminoglycan synthesis together with impaired glycanation of the small chondroitin/dermatan sulfate proteoglycan decorin, confirming β3GalT6 loss of function. Dermal electron microcopy disclosed abnormalities in collagen fibril organization, in line with the important regulatory role of decorin in this process. A strong reduction in heparan sulfate level was also observed, indicating that β3GalT6 deficiency alters synthesis of both main types of glycosaminoglycans. In vitro wound healing assay revealed a significant delay in fibroblasts from two index individuals, pointing to a role for glycosaminoglycan defect in impaired wound repair in vivo. Our study emphasizes a crucial role for β3GalT6 in multiple major developmental and pathophysiological processes.     

Recurrent Muscle Weakness with Rhabdomyolysis,Metabolic Crises,and Cardiac Arrhythmia Due to Bi-allelic TANGO2 Mutations

The underlying genetic etiology of rhabdomyolysis remains elusive in a significant fraction of individuals presenting with recurrent metabolic crises and muscle weakness. Using exome sequencing, we identified bi-allelic mutations in TANGO2 encoding transport and Golgi organization 2 homolog (Drosophila) in 12 subjects with episodic rhabdomyolysis, hypoglycemia, hyperammonemia, and susceptibility to life-threatening cardiac tachyarrhythmias. A recurrent homozygous c.460G>A (p.Gly154Arg) mutation was found in four unrelated individuals of Hispanic/Latino origin, and a homozygous ∼34 kb deletion affecting exons 3–9 was observed in two families of European ancestry. One individual of mixed Hispanic/European descent was found to be compound heterozygous for c.460G>A (p.Gly154Arg) and the deletion of exons 3–9. Additionally, a homozygous exons 4–6 deletion was identified in a consanguineous Middle Eastern Arab family. No homozygotes have been reported for these changes in control databases. Fibroblasts derived from a subject with the recurrent c.460G>A (p.Gly154Arg) mutation showed evidence of increased endoplasmic reticulum stress and a reduction in Golgi volume density in comparison to control. Our results show that the c.460G>A (p.Gly154Arg) mutation and the exons 3–9 heterozygous deletion in TANGO2 are recurrent pathogenic alleles present in the Latino/Hispanic and European populations, respectively, causing considerable morbidity in the homozygotes in these populations.     

A novel Gly137Asp MPZ mutation in a Charcot-Marie-Tooth disease type 1B family

Charcot-Marie-Tooth disease (CMT) is a clinically and genetically heterogeneous peripheral neuropathy. Myelin protein zero (MPZ) is a major myelin protein of the peripheral nerve and mutations in its gene cause a wide phenotypic spectrum including CMT1B, CMT2I, CMT2J and Dejerine-Sottas syndrome. The objective of this study was to find the causative mutation in a Korean large autosomal dominant demyelinating CMT family (FC240). Clinical disabilities were measured by a functional disability scale (FDS), a CMT neuropathy score (CMTNS), and a 9-hole peg test (9-HPT). Mutational analysis of the FC240 family revealed a novel c.410G>A (Gly137Asp) mutation in the MPZ gene. The mutation was completely co-segregated with affected members in the family, and was not found in controls. The clinical features and electrophysiological findings were compatible with CMT1B. Clinical symptoms revealed phenotypic diversities among family members and generations within the same family. In addition, the present patients with Gly137Asp mutation showed earlier age at onset and slow progression than the reported patient with Gly137Ser. Therefore, it seems that there were wide phenotypic variations within the same family harboring Gly137Asp mutation, and between Gly137Asp and Gly137Ser mutations.     

Drosophila GoLoco-Protein Pins Is a Target of Gαo-mediated G Protein–coupled Receptor Signaling

G protein–coupled receptors (GPCRs) transduce their signals through trimeric G proteins, inducing guanine nucleotide exchange on their Gα-subunits; the resulting Gα-GTP transmits the signal further inside the cell. GoLoco domains present in many proteins play important roles in multiple trimeric G protein–dependent activities, physically binding Gα-subunits of the Gα_i/o class. In most cases GoLoco binds exclusively to the GDP-loaded form of the Gα-subunits. Here we demonstrate that the poly-GoLoco–containing protein Pins of Drosophila can bind to both GDP- and GTP-forms of Drosophila Gα_o. We identify Pins GoLoco domain 1 as necessary and sufficient for this unusual interaction with Gα_o-GTP. We further pinpoint a lysine residue located centrally in this domain as necessary for the interaction. Our studies thus identify Drosophila Pins as a target of Gα_o-mediated GPCR receptor signaling, e.g., in the context of the nervous system development, where Gα_o acts downstream from Frizzled and redundantly with Gα_i to control the asymmetry of cell divisions.     

KRAS mutations in colorectal cancer from Tunisia: relationships with clinicopathologic variables and data on TP53 mutations and microsatellite instability

Mutations in KRAS gene are among the critical transforming alterations occurring during CRC tumorigenesis. Here we screened 51 primary CRC tumors from Tunisia for mutations in KRAS (codons 12 and 13) using PCR-direct sequencing. Our aim was to analyze tumor mutation frequencies and spectra in Tunisian patients with CRC. KRAS status and mutation site/type were than correlated with familial and clinicopathologic variables and data on TP53 mutations and nuclear protein accumulation and microsatellite instability (MSI). A KRAS somatic mutation has been detected in the CRC tumor of 31.5 % (16/51) of the patients. 81.2 % had a single mutation at codon 12 and 23 % had a single mutation at codon 13. The most common single mutation (50 %) was a G>A transition in codon 12 (c.35G>A; p.Gly12Asp). 81.25 % of the KRAS mutations were transitions and 23 % were transversions. All the mutations in codon 13 were a c.38G>A transition, whereas both G>A transitions and G>T and G>C transversions were found in codon 12. The mutation spectrum was different between MSS and MSI-H tumors and more varied mutations have been detected in MSS tumors. Some amino acid changes were detected only in MSS tumors, i.e. p.Gly12Ser, p.Gly12Cys and p.Gly12Ala. Whereas, the KRAS mutation p.Gly13Asp have been detected only in MSI-H. 43.75 % of the patients harboured combined mutations in KRAS and TP53 genes and the tumor of 71.42 % of them showed TP53 overexpression. In conclusion, the frequency and types of KRAS mutations were as reported for non-Tunisian patients. However, no significant associations have been detected between KRAS mutations and clinicopathologic variables and MSI in Tunisian patients as previously reported.     

The Z Mutation Alters the Global Structural Dynamics of α1-Antitrypsin

α₁-Antitrypsin (α₁AT) deficiency, the most common serpinopathy, results in both emphysema and liver disease. Over 90% of all clinical cases of α₁AT deficiency are caused by the Z variant in which Glu342, located at the top of s5A, is replaced by a Lys which results in polymerization both in vivo and in vitro. The Glu342Lys mutation removes a salt bridge and a hydrogen bond but does not effect the thermodynamic stability of Z α₁AT compared to the wild type protein, M α₁AT, and so it is unclear why Z α₁AT has an increased polymerization propensity. We speculated that the loss of these interactions would make the native state of Z α₁AT more dynamic than M α₁AT and that this change renders the protein more polymerization prone. We have used hydrogen/deuterium exchange combined with mass spectrometry (HXMS) to determine the structural and dynamic differences between native Z and M α₁AT to reveal the molecular basis of Z α₁AT polymerization. Our HXMS data shows that the Z mutation significantly perturbs the region around the site of mutation. Strikingly the Z mutation also alters the dynamics of regions distant to the mutation such as the B, D and I helices and specific regions of each β-sheet. These changes in global dynamics may lead to an increase in the likelihood of Z α₁AT sampling a polymerogenic structure thereby causing disease.     

Regulator of G Protein Signaling 2 (RGS2) and RGS4 Form Distinct G Protein-Dependent Complexes with Protease Activated-Receptor 1 (PAR1) in Live Cells

Protease-activated receptor 1 (PAR1) is a G-protein coupled receptor (GPCR) that is activated by natural proteases to regulate many physiological actions. We previously reported that PAR1 couples to G_i, G_q and G₁₂ to activate linked signaling pathways. Regulators of G protein signaling (RGS) proteins serve as GTPase activating proteins to inhibit GPCR/G protein signaling. Some RGS proteins interact directly with certain GPCRs to modulate their signals, though cellular mechanisms dictating selective RGS/GPCR coupling are poorly understood. Here, using bioluminescence resonance energy transfer (BRET), we tested whether RGS2 and RGS4 bind to PAR1 in live COS-7 cells to regulate PAR1/Gα-mediated signaling. We report that PAR1 selectively interacts with either RGS2 or RGS4 in a G protein-dependent manner. Very little BRET activity is observed between PAR1-Venus (PAR1-Ven) and either RGS2-Luciferase (RGS2-Luc) or RGS4-Luc in the absence of Gα. However, in the presence of specific Gα subunits, BRET activity was markedly enhanced between PAR1-RGS2 by Gα_q/11, and PAR1-RGS4 by Gα_o, but not by other Gα subunits. Gα_q/11-YFP/RGS2-Luc BRET activity is promoted by PAR1 and is markedly enhanced by agonist (TFLLR) stimulation. However, PAR1-Ven/RGS-Luc BRET activity was blocked by a PAR1 mutant (R205A) that eliminates PAR1-G_q/11 coupling. The purified intracellular third loop of PAR1 binds directly to purified His-RGS2 or His-RGS4. In cells, RGS2 and RGS4 inhibited PAR1/Gα-mediated calcium and MAPK/ERK signaling, respectively, but not RhoA signaling. Our findings indicate that RGS2 and RGS4 interact directly with PAR1 in Gα-dependent manner to modulate PAR1/Gα-mediated signaling, and highlight a cellular mechanism for selective GPCR/G protein/RGS coupling.     

The interaction of Gα13 with integrin β1 mediates cell migration by dynamic regulation of RhoA

Heterotrimeric G protein Gα₁₃ is known to transmit G protein–coupled receptor (GPCR) signals leading to activation of RhoA and plays a role in cell migration. The mechanism underlying the role of Gα₁₃ in cell migration, however, remains unclear. Recently we found that Gα₁₃ interacts with the cytoplasmic domain of integrin β₃ subunits in platelets via a conserved ExE motif. Here we show that a similar direct interaction between Gα₁₃ and the cytoplasmic domain of the integrin β₁ subunit plays a critical role in β₁-dependent cell migration. Point mutation of either glutamic acid in the Gα₁₃-binding ⁷⁶⁷EKE motif in β₁ or treatment with a peptide derived from the Gα₁₃-binding sequence of β₁ abolished Gα₁₃–β₁ interaction and inhibited β₁ integrin–dependent cell spreading and migration. We further show that the Gα₁₃-β₁ interaction mediates β₁ integrin–dependent Src activation and transient RhoA inhibition during initial cell adhesion, which is in contrast to the role of Gα₁₃ in mediating GPCR-dependent RhoA activation. These data indicate that Gα₁₃ plays dynamic roles in both stimulating RhoA via a GPCR pathway and inhibiting RhoA via an integrin signaling pathway. This dynamic regulation of RhoA activity is critical for cell migration on β₁ integrin ligands.     

Characterization of Candida albicans RNA triphosphatase and mutational analysis of its active site

The RNA triphosphatase component (CaCet1p) of the mRNA capping apparatus of the pathogenic fungus Candida albicans differs mechanistically and structurally from the RNA triphosphatase of mammals. Hence, CaCet1p is an attractive antifungal target. Here we identify a C-terminal catalytic domain of CaCet1p from residue 257 to 520 and characterize a manganese-dependent and cobalt-dependent NTPase activity intrinsic to CaCet1p. The NTPase can be exploited to screen in vitro for inhibitors. The amino acids that comprise the active site of CaCet1p were identified by alanine-scanning mutagenesis, which was guided by the crystal structure of the homologous RNA triphosphatase from Saccharomyces cerevisiae (Cet1p). Thirteen residues required for the phosphohydrolase activity of CaCet1p (Glu287, Glu289, Asp363, Arg379, Lys396, Glu420, Arg441, Lys443, Arg445, Asp458, Glu472, Glu474 and Glu476) are located within the hydrophilic interior of an eight-strand β barrel of Cet1p. Each of the eight strands contributes at least one essential amino acid. The essential CaCet1p residues include all of the side chains that coordinate manganese and sulfate (i.e., γ phosphate) in the Cet1p product complex. These results suggest that the active site structure and catalytic mechanism are conserved among fungal RNA triphosphatases.     

Detection of G Protein-selective G Protein-coupled Receptor (GPCR) Conformations in Live Cells

Although several recent studies have reported that GPCRs adopt multiple conformations, it remains unclear how subtle conformational changes are translated into divergent downstream responses. In this study, we report on a novel class of FRET-based sensors that can detect the ligand/mutagenic stabilization of GPCR conformations that promote interactions with G proteins in live cells. These sensors rely on the well characterized interaction between a GPCR and the C terminus of a Gα subunit. We use these sensors to elucidate the influence of the highly conserved (E/D)RY motif on GPCR conformation. Specifically, Glu/Asp but not Arg mutants of the (E/D)RY motif are known to enhance basal GPCR signaling. Hence, it is unclear whether ionic interactions formed by the (E/D)RY motif (ionic lock) are necessary to stabilize basal GPCR states. We find that mutagenesis of the β2-AR (E/D)RY ionic lock enhances interaction with G_s. However, only Glu/Asp but not Arg mutants increase G protein activation. In contrast, mutagenesis of the opsin (E/D)RY ionic lock does not alter its interaction with transducin. Instead, opsin-specific ionic interactions centered on residue Lys-296 are both necessary and sufficient to promote interactions with transducin. Effective suppression of β2-AR basal activity by inverse agonist ICI 118,551 requires ionic interactions formed by the (E/D)RY motif. In contrast, the inverse agonist metoprolol suppresses interactions with G_s and promotes G_i binding, with concomitant pertussis toxin-sensitive inhibition of adenylyl cyclase activity. Taken together, these studies validate the use of the new FRET sensors while revealing distinct structural mechanisms for ligand-dependent GPCR function.     

Pivotal Role of Extended Linker 2 in the Activation of Gα by G Protein-coupled Receptor

G protein-coupled receptors (GPCRs) relay extracellular signals mainly to heterotrimeric G-proteins (Gαβγ) and they are the most successful drug targets. The mechanisms of G-protein activation by GPCRs are not well understood. Previous studies have revealed a signal relay route from a GPCR via the C-terminal α5-helix of Gα to the guanine nucleotide-binding pocket. Recent structural and biophysical studies uncover a role for the opening or rotating of the α-helical domain of Gα during the activation of Gα by a GPCR. Here we show that β-adrenergic receptors activate eight Gα_s mutant proteins (from a screen of 66 Gα_s mutants) that are unable to bind Gβγ subunits in cells. Five of these eight mutants are in the αF/Linker 2/β2 hinge region (extended Linker 2) that connects the Ras-like GTPase domain and the α-helical domain of Gα_s. This extended Linker 2 is the target site of a natural product inhibitor of G_q. Our data show that the extended Linker 2 is critical for Gα activation by GPCRs. We propose that a GPCR via its intracellular loop 2 directly interacts with the β₂/β₃ loop of Gα to communicate to Linker 2, resulting in the opening and closing of the α-helical domain and the release of GDP during G-protein activation.