DOCSLIB.ORG

GNAT1 Associated with Autosomal Recessive Congenital Stationary Night Blindness

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
GNAT1 Associated with Autosomal Recessive Congenital Stationary Night Blindness Retina GNAT1 Associated with Autosomal Recessive Congenital Stationary Night Blindness Muhammad Asif Naeem,1,2 Venkata R. M. Chavali,2,3 Shahbaz Ali,1 Muhammad Iqbal,1 Saima Riazuddin,1,4,5 Shaheen N. Khan,1 Tayyab Husnain,1 Paul A. Sieving,6 Radha Ayyagari,3 Sheikh Riazuddin,1,7 J. Fielding Hejtmancik,6,8 and S. Amer Riazuddin1,8,9 PURPOSE. Congenital stationary night blindness is a nonprogres- itance, and was not present in 192 ethnically matched control sive retinal disorder manifesting as impaired night vision and is chromosomes. Expression analysis suggested that Gnat1 is ex- generally associated with other ocular symptoms, such as nys- pressed at approximately postnatal day (P)7 and is predomi- tagmus, myopia, and strabismus. This study was conducted to nantly expressed in the retina. further investigate the genetic basis of CSNB in a consanguin- CONCLUSIONS. These data suggest that a homozygous missense eous Pakistani family. mutation in GNAT1 is associated with autosomal recessive METHODS. A consanguineous family with multiple individuals stationary night blindness. (Invest Ophthalmol Vis Sci. 2012; manifesting cardinal symptoms of congenital stationary night 53:1353–1361) DOI:10.1167/iovs.11-8026 blindness was ascertained. All family members underwent de- tailed ophthalmic examination, including fundus photographic ongenital stationary night blindness (CSNB) is a clinically examination and electroretinography. Blood samples were col- Cheterogeneous group of nonprogressive retinal disorders, lected and genomic DNA was extracted. Exclusion and ge- characterized by impaired night vision, decreased visual acuity, nome-wide linkage analyses were completed and two-point nystagmus, myopia, and strabismus.1 CSNB can be classified LOD scores were calculated. Bidirectional sequencing of into two groups based on the electroretinographic recordings GNAT1 was completed, and quantitative expression of Gnat1 that exhibit waveforms in response to flashes of light that transcript levels were investigated in ocular tissues at different correspond to changes in the polarization of the photoreceptor postnatal intervals. and bipolar cells.2,3 The Schubert-Bornschein type is charac- RESULTS. The results of ophthalmic examinations were sugges- terized by an electronegative electroretinogram (ERG) at the tive of early-onset stationary night blindness with no extraoc- scotopic bright flash, in which the amplitude of the b-wave is ular anomalies. The genome-wide scan localized the critical smaller than that of the a-wave,2 while the Riggs type is defined interval to chromosome 3, region p22.1-p14.3, with maximum by proportionally reduced a- and b-waves.3 The Schubert-Born- two-point LOD scores of 3.09 at ␪ ϭ 0, flanked by markers schein- and Riggs-type CSNB patients not only differ electro- D3S3522 and D3S1289. Subsequently, a missense mutation in physiologically, but manifest different clinical characteristics. GNAT1, p.D129G, was identified, which segregated within the Decreased visual acuity, myopia, and nystagmus can be asso- family, consistent with an autosomal recessive mode of inher- ciated with Schubert-Bornschein CSNB, whereas patients with Riggs-type CSNB have visual acuity within normal range with no symptoms of myopia and/or nystagmus.1 Familial cases of CSNB with autosomal dominant, autosomal From the 1National Centre of Excellence in Molecular Biology, University of the Punjab, Lahore, Pakistan; the 3Shiley Eye Center, recessive as well as X-linked inheritance have been reported. University of California San Diego, La Jolla, California; the Divisions of Mutations in RHO, PDE6B, and GNAT1 have been associated 4Pediatric Otolaryngology Head and Neck Surgery and 5Ophthalmol- with autosomal dominant CSNB, and patients with mutations ogy, Cincinnati Children Hospital Research Foundation, Cincinnati, in these genes exhibit the Riggs- or Schubert-Bornschein-type Ohio; 6Ophthalmic Genetics and Visual Function Branch, National Eye ERG.4–10 The Schubert-Bornschein form can be further subdi- Institute, National Institutes of Health, Bethesda Maryland; the 7Allama vided into complete and incomplete types. Complete CSNB is Iqbal Medical College, University of Health Sciences, Lahore, Pakistan; characterized by reduced rod b-wave response, whereas the 9 and The Wilmer Eye Institute, Johns Hopkins University School of incomplete CSNB is characterized by both a reduced rod b- Medicine, Baltimore, Maryland. 2 wave and substantially reduced flicker cone responses, due to These authors contributed equally to the work presented here 11 and should therefore be regarded as equivalent first authors. both ON and OFF bipolar cell dysfunction. Mutations in 8 GRM6 and TRPM1 lead to autosomal recessive CSNB, whereas These authors contributed equally to the work presented here 12–18 and should therefore be regarded as equivalent senior authors. mutations in NYX to X-linked complete CSNB. Mutations Supported in part by the Higher Education Commission (HEC) and in CABP4 have been associated with autosomal recessive the Ministry of Science and Technology, Islamabad, Pakistan, and by CSNB and mutations in CACNA1F with X-linked incomplete National Eye Institute Grant R01EY021237-01 (RA, SAR). CSNB.19–21 Recently, SLC24A1 was implicated in the patho- Submitted for publication June 10, 2011; revised October 31, genesis of autosomal recessive CSNB with Riggs-type ERG 2011; accepted November 4, 2011. response.22 Disclosure: M.A. Naeem, None; V.R.M. Chavali, None; S. Ali, Transducin is a trimeric G protein that is expressed in the None; M. Iqbal, None; Sa. Riazuddin, None; S.N. Khan, None; T. 23 ␣ Husnain, None; P.A. Sieving, None; R. Ayyagari, None; Sh. Riazuddin, retina and consists of three subunits. The -subunit binds None; J.F. Hejtmancik, None; S.A. Riazuddin, None GTP and activates cyclic GMP phosphodiesterase (PDE), Corresponding author: S. Amer Riazuddin, The Wilmer Eye Insti- whereas the ␤ and ␥ subunits form a complex that is necessary 24 tute, Johns Hopkins University School of Medicine, 600 N. Wolfe to interact with rhodopsin. The ␣-subunit expressed in the Street, Maumenee 809A, Baltimore MD 21287; [email protected]. rod cells is encoded by GNAT1, whereas ␣-subunit expressed Investigative Ophthalmology & Visual Science, March 2012, Vol. 53, No. 3 Copyright 2012 The Association for Research in Vision and Ophthalmology, Inc. 1353 Downloaded from jov.arvojournals.org on 09/28/2021 1354 Naeem et al. IOVS, March 2012, Vol. 53, No. 3 in the cone cells is encoded by GNAT2.25,26 Transducin is age scan localized the disease phenotype to chromosome 3, activated by conformational changes in rhodopsin due to the region p22.1-p14.3, harboring GNAT1, a gene previously asso- absorption of light, which causes a GDP bound ␣ subunit to be ciated with autosomal dominant CSNB. Bidirectional sequenc- exchanged with GTP and subsequent dissociation from the ing identified a homozygous missense mutation in GNAT1 that ␤-gamma complex.27,28 segregated with the disease phenotype in the family and was Previously, a missense mutation c.113GϾA was identified in not present in ethnically matched control chromosomes. To the Nougaret form of autosomal dominant CSNB.9 This muta- the best of our knowledge, this is the first report to implicate tion (Gly38Arg) affects part of small loop of ␣ subunit of rod GNAT1 in the pathogenesis of autosomal recessive CSNB. transducin and is believed to affect GTPase activity.9 Recently, Szabo et al.10 identified a heterozygous mutation; c.598CϾG, in exon 6 of GNAT1 in individuals affected with autosomal MATERIALS AND METHODS dominant CSNB. This mutation (Gln200Glu) resides in a do- main that plays an important role in binding and hydrolyzing Clinical Assessment GTP, and the ERG recordings were suggestive of a nonprogres- A consanguineous Pakistani family with four affected individuals with sive presynaptic defect in rod phototransduction.10 a history of night blindness was recruited to participate in study to Here, we report a consanguineous family with multiple investigate autosomal recessive CSNB. Institutional review board (IRB) individuals diagnosed with CSNB that segregated within the approval for this study was obtained from the National Centre of family in an autosomal recessive fashion. A genome-wide link- Excellence in Molecular Biology and the National Eye Institute. The FIGURE 1. Pedigree of PKRP130 with a haplotype formed from alleles of the short arm of chromosome 3 microsatel- lite markers. Alleles forming the risk hap- lotype (black), alleles cosegregating with CSNB but not showing homozygos- ity (gray), and alleles not cosegregating with CSNB (white) are shown. Squares: males; circles: females; filled symbols: affected individuals; double line be- tween individuals: consanguinity; and diagonal line through a symbol: deceased member. Downloaded from jov.arvojournals.org on 09/28/2021 IOVS, March 2012, Vol. 53, No. 3 GNAT1-Associated Stationary Night Blindness 1355 participating subjects gave informed written consent consistent with Applied Biosystems, Inc. [ABI], Foster City, CA) having an average the tenets of the Declaration of Helsinki. Funduscopy was performed spacing of 10 centimorgans (cM). Multiplex polymerase chain reac- at Layton Rehmatullah Benevolent Trust (LRBT) Hospital (Lahore, Pak- tions were performed, as follows (9700 PCR System; ABI): Each reac- istan). Electroretinography (ERG) measurements were recorded by tion was performed in a 5-␮L mixture containing 40 ng genomic DNA, using equipment manufactured by LKC (Gaithersburg,
Load more

Recommended publications
  • Predicting Coupling Probabilities of G-Protein Coupled Receptors Gurdeep Singh1,2,†, Asuka Inoue3,*,†, J
    Published online 30 May 2019 Nucleic Acids Research, 2019, Vol. 47, Web Server issue W395–W401 doi: 10.1093/nar/gkz392 PRECOG: PREdicting COupling probabilities of G-protein coupled receptors Gurdeep Singh1,2,†, Asuka Inoue3,*,†, J. Silvio Gutkind4, Robert B. Russell1,2,* and Francesco Raimondi1,2,* 1CellNetworks, Bioquant, Heidelberg University, Im Neuenheimer Feld 267, 69120 Heidelberg, Germany, 2Biochemie Zentrum Heidelberg (BZH), Heidelberg University, Im Neuenheimer Feld 328, 69120 Heidelberg, Germany, 3Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, Miyagi 980-8578, Japan and 4Department of Pharmacology and Moores Cancer Center, University of California, San Diego, La Jolla, CA 92093, USA Received February 10, 2019; Revised April 13, 2019; Editorial Decision April 24, 2019; Accepted May 01, 2019 ABSTRACT great use in tinkering with signalling pathways in living sys- tems (5). G-protein coupled receptors (GPCRs) control multi- Ligand binding to GPCRs induces conformational ple physiological states by transducing a multitude changes that lead to binding and activation of G-proteins of extracellular stimuli into the cell via coupling to situated on the inner cell membrane. Most of mammalian intra-cellular heterotrimeric G-proteins. Deciphering GPCRs couple with more than one G-protein giving each which G-proteins couple to each of the hundreds receptor a distinct coupling profile (6) and thus specific of GPCRs present in a typical eukaryotic organism downstream cellular responses. Determining these coupling is therefore critical to understand signalling. Here, profiles is critical to understand GPCR biology and phar- we present PRECOG (precog.russelllab.org): a web- macology. Despite decades of research and hundreds of ob- server for predicting GPCR coupling, which allows served interactions, coupling information is still missing for users to: (i) predict coupling probabilities for GPCRs many receptors and sequence determinants of coupling- specificity are still largely unknown.
    [Show full text]
  • G-Protein ␤␥-Complex Is Crucial for Efficient Signal Amplification in Vision
    The Journal of Neuroscience, June 1, 2011 • 31(22):8067–8077 • 8067 Cellular/Molecular G-Protein ␤␥-Complex Is Crucial for Efficient Signal Amplification in Vision Alexander V. Kolesnikov,1 Loryn Rikimaru,2 Anne K. Hennig,1 Peter D. Lukasiewicz,1 Steven J. Fliesler,4,5,6,7 Victor I. Govardovskii,8 Vladimir J. Kefalov,1 and Oleg G. Kisselev2,3 1Department of Ophthalmology and Visual Sciences, Washington University School of Medicine, St. Louis, Missouri 63110, Departments of 2Ophthalmology and 3Biochemistry and Molecular Biology, Saint Louis University School of Medicine, Saint Louis, Missouri 63104, 4Research Service, Veterans Administration Western New York Healthcare System, and Departments of 5Ophthalmology (Ross Eye Institute) and 6Biochemistry, University at Buffalo/The State University of New York (SUNY), and 7SUNY Eye Institute, Buffalo, New York 14215, and 8Sechenov Institute for Evolutionary Physiology and Biochemistry, Russian Academy of Sciences, Saint Petersburg 194223, Russia A fundamental question of cell signaling biology is how faint external signals produce robust physiological responses. One universal mechanism relies on signal amplification via intracellular cascades mediated by heterotrimeric G-proteins. This high amplification system allows retinal rod photoreceptors to detect single photons of light. Although much is now known about the role of the ␣-subunit of the rod-specific G-protein transducin in phototransduction, the physiological function of the auxiliary ␤␥-complex in this process remains a mystery. Here, we show that elimination of the transducin ␥-subunit drastically reduces signal amplification in intact mouse rods. The consequence is a striking decline in rod visual sensitivity and severe impairment of nocturnal vision. Our findings demonstrate that transducin ␤␥-complex controls signal amplification of the rod phototransduction cascade and is critical for the ability of rod photoreceptors to function in low light conditions.
    [Show full text]
  • G Protein Alpha Inhibitor 1 (GNAI1) Rabbit Polyclonal Antibody Product Data
    OriGene Technologies, Inc. 9620 Medical Center Drive, Ste 200 Rockville, MD 20850, US Phone: +1-888-267-4436 [email protected] EU: [email protected] CN: [email protected] Product datasheet for TA335036 G protein alpha inhibitor 1 (GNAI1) Rabbit Polyclonal Antibody Product data: Product Type: Primary Antibodies Applications: WB Recommended Dilution: WB Reactivity: Human, Mouse, Rat Host: Rabbit Isotype: IgG Clonality: Polyclonal Immunogen: The immunogen for anti-GNAI1 antibody: synthetic peptide directed towards the middle region of human GNAI1. Synthetic peptide located within the following region: YQLNDSAAYYLNDLDRIAQPNYIPTQQDVLRTRVKTTGIVETHFTFKDLH Formulation: Liquid. Purified antibody supplied in 1x PBS buffer with 0.09% (w/v) sodium azide and 2% sucrose. Note that this product is shipped as lyophilized powder to China customers. Purification: Affinity Purified Conjugation: Unconjugated Storage: Store at -20°C as received. Stability: Stable for 12 months from date of receipt. Predicted Protein Size: 40 kDa Gene Name: G protein subunit alpha i1 Database Link: NP_002060 Entrez Gene 14677 MouseEntrez Gene 25686 RatEntrez Gene 2770 Human P63096 This product is to be used for laboratory only. Not for diagnostic or therapeutic use. View online » ©2021 OriGene Technologies, Inc., 9620 Medical Center Drive, Ste 200, Rockville, MD 20850, US 1 / 4 G protein alpha inhibitor 1 (GNAI1) Rabbit Polyclonal Antibody – TA335036 Background: Guanine nucleotide-binding proteins (G proteins) form a large family of signal-transducing molecules. They are found as heterotrimers made up of alpha, beta, and gamma subunits. Members of the G protein family have been characterized most extensively on the basis of the alpha subunit, which binds guanine nucleotide, is capable of hydrolyzing GTP, and interacts with specific receptor and effector molecules.
    [Show full text]
  • GNAT1 (NM 144499) Human Tagged ORF Clone Product Data
    OriGene Technologies, Inc. 9620 Medical Center Drive, Ste 200 Rockville, MD 20850, US Phone: +1-888-267-4436 [email protected] EU: [email protected] CN: [email protected] Product datasheet for RC221246 GNAT1 (NM_144499) Human Tagged ORF Clone Product data: Product Type: Expression Plasmids Product Name: GNAT1 (NM_144499) Human Tagged ORF Clone Tag: Myc-DDK Symbol: GNAT1 Synonyms: CSNB1G; CSNBAD3; GBT1; GNATR Vector: pCMV6-Entry (PS100001) E. coli Selection: Kanamycin (25 ug/mL) Cell Selection: Neomycin ORF Nucleotide >RC221246 representing NM_144499 Sequence: Red=Cloning site Blue=ORF Green=Tags(s) TTTTGTAATACGACTCACTATAGGGCGGCCGGGAATTCGTCGACTGGATCCGGTACCGAGGAGATCTGCC GCCGCGATCGCC ATGGGGGCTGGGGCCAGTGCTGAGGAGAAGCACTCCAGGGAGCTGGAAAAGAAGCTGAAAGAGGACGCTG AGAAGGATGCTCGAACCGTGAAGCTGCTGCTTCTGGGTGCCGGTGAGTCCGGGAAGAGCACCATCGTCAA GCAGATGAAGATTATCCACCAGGACGGGTACTCGCTGGAAGAGTGCCTCGAGTTTATCGCCATCATCTAC GGCAACACGTTGCAGTCCATCCTGGCCATCGTACGCGCCATGACCACACTCAACATCCAGTACGGAGACT CTGCACGCCAGGACGACGCCCGGAAGCTGATGCACATGGCAGACACTATCGAGGAGGGCACGATGCCCAA GGAGATGTCGGACATCATCCAGCGGCTGTGGAAGGACTCCGGTATCCAGGCCTGTTTTGAGCGCGCCTCG GAGTACCAGCTCAACGACTCGGCGGGCTACTACCTCTCCGACCTGGAGCGCCTGGTAACCCCGGGCTACG TGCCCACCGAGCAGGACGTGCTGCGCTCGCGAGTCAAGACCACTGGCATCATCGAGACGCAGTTCTCCTT CAAGGATCTCAACTTCCGGATGTTCGATGTGGGCGGGCAGCGCTCGGAGCGCAAGAAGTGGATCCACTGC TTCGAGGGCGTGACCTGCATCATCTTCATCGCGGCGCTGAGCGCCTACGACATGGTGCTAGTGGAGGACG ACGAAGTGAACCGCATGCACGAGAGCCTGCACCTGTTCAACAGCATCTGCAACCACCGCTACTTCGCCAC GACGTCCATCGTGCTCTTCCTTAACAAGAAGGACGTCTTCTTCGAGAAGATCAAGAAGGCGCACCTCAGC ATCTGTTTCCCGGACTACGATGGACCCAACACCTACGAGGACGCCGGCAACTACATCAAGGTGCAGTTCC
    [Show full text]
  • G Protein-Coupled Receptors
    G PROTEIN-COUPLED RECEPTORS Overview:- The completion of the Human Genome Project allowed the identification of a large family of proteins with a common motif of seven groups of 20-24 hydrophobic amino acids arranged as α-helices. Approximately 800 of these seven transmembrane (7TM) receptors have been identified of which over 300 are non-olfactory receptors (see Frederikson et al., 2003; Lagerstrom and Schioth, 2008). Subdivision on the basis of sequence homology allows the definition of rhodopsin, secretin, adhesion, glutamate and Frizzled receptor families. NC-IUPHAR recognizes Classes A, B, and C, which equate to the rhodopsin, secretin, and glutamate receptor families. The nomenclature of 7TM receptors is commonly used interchangeably with G protein-coupled receptors (GPCR), although the former nomenclature recognises signalling of 7TM receptors through pathways not involving G proteins. For example, adiponectin and membrane progestin receptors have some sequence homology to 7TM receptors but signal independently of G-proteins and appear to reside in membranes in an inverted fashion compared to conventional GPCR. Additionally, the NPR-C natriuretic peptide receptor has a single transmembrane domain structure, but appears to couple to G proteins to generate cellular responses. The 300+ non-olfactory GPCR are the targets for the majority of drugs in clinical usage (Overington et al., 2006), although only a minority of these receptors are exploited therapeutically. Signalling through GPCR is enacted by the activation of heterotrimeric GTP-binding proteins (G proteins), made up of α, β and γ subunits, where the α and βγ subunits are responsible for signalling. The α subunit (tabulated below) allows definition of one series of signalling cascades and allows grouping of GPCRs to suggest common cellular, tissue and behavioural responses.
    [Show full text]
  • ANALYSIS Doi:10.1038/Nature14663
    ANALYSIS doi:10.1038/nature14663 Universal allosteric mechanism for Ga activation by GPCRs Tilman Flock1, Charles N. J. Ravarani1*, Dawei Sun2,3*, A. J. Venkatakrishnan1{, Melis Kayikci1, Christopher G. Tate1, Dmitry B. Veprintsev2,3 & M. Madan Babu1 G protein-coupled receptors (GPCRs) allosterically activate heterotrimeric G proteins and trigger GDP release. Given that there are 800 human GPCRs and 16 different Ga genes, this raises the question of whether a universal allosteric mechanism governs Ga activation. Here we show that different GPCRs interact with and activate Ga proteins through a highly conserved mechanism. Comparison of Ga with the small G protein Ras reveals how the evolution of short segments that undergo disorder-to-order transitions can decouple regions important for allosteric activation from receptor binding specificity. This might explain how the GPCR–Ga system diversified rapidly, while conserving the allosteric activation mechanism. proteins bind guanine nucleotides and act as molecular switches almost 30 A˚ away from the GDP binding region5 and allosterically trig- in a number of signalling pathways by interconverting between ger GDP release to activate them. 1,2 G a GDP-bound inactive and a GTP-bound active state . They The high-resolution structure of the Gas-bound b2-adrenergic recep- 3 5 consist of two major classes: monomeric small G proteins and hetero- tor (b2AR) provided crucial insights into the receptor–G protein inter- trimeric G proteins4. While small G proteins and the a-subunit (Ga)of face and conformational changes in Ga upon receptor binding6,7. Recent 6 8 heterotrimeric G proteins both contain a GTPase domain (G-domain), studies described dynamic regions in Gas and Gai , the importance of ˚ Ga contains an additional helical domain (H-domain) and also forms a displacement of helix 5 (H5) of Gas and Gat by up to 6 A into the complex with the Gb and Gc subunits.
    [Show full text]
  • Phototransduction Cascade Activation Steps
    1 ORIGINAL RESEARCH ARTICLE Evolution of the vertebrate phototransduction cascade activation steps Trevor D. Lamb1 and David M. Hunt2,3 1Eccles Institute of Neuroscience, John Curtin School of Medical Research, The Australian National University, ACT 2600, Australia, and 2The Lions Eye Institute and 3School of Biological Sciences, The University of Western Australia, WA 6009, Australia. Correspondence: Professor Trevor Lamb <[email protected]> Keywords: Evolution; Phototransduction; Transducin; Phosphodiesterase; Cyclic nucleotide- gated channel; Opsin ABSTRACT We examine the molecular phylogeny of the proteins underlying the activation steps of vertebrate phototransduction, for both agnathan and jawed vertebrate taxa. We expand the number of taxa analysed and we update the alignment and tree building methodology from a previous analysis. For each of the four primary components (the G-protein transducin alpha subunit, GαT, the cyclic GMP phosphodiesterase, PDE6, and the alpha and beta subunits of the cGMP-gated ion channel, CNGC), the phylogenies appear consistent with expansion from an ancestral proto-vertebrate cascade during two rounds of whole-genome duplication followed by divergence of the agnathan and jawed vertebrate lineages. In each case, we consider possible scenarios for the underlying gene duplications and losses, and we apply relevant constraints to the tree construction. From tests of the topology of the resulting trees, we obtain a scenario for the expansion of each component during 2R that accurately fits the observations. Similar analysis of the visual opsins indicates that the only expansion to have occurred during 2R was the formation of Rh1 and Rh2. Finally, we propose a hypothetical scenario for the conversion of an ancestral chordate cascade into the proto-vertebrate phototransduction cascade, prior to whole-genome duplication.
    [Show full text]
  • The Specificity of Downstream Signaling for A1 and A2AR
    biomedicines Article The Specificity of Downstream Signaling for A1 and A2AR Does Not Depend on the C-Terminus, Despite the Importance of This Domain in Downstream Signaling Strength Abhinav R. Jain 1 , Claire McGraw 1 and Anne S. Robinson 1,2,* 1 Department of Chemical and Biomolecular Engineering, Tulane University, New Orleans, LA 70118, USA; [email protected] (A.R.J.); [email protected] (C.M.) 2 Department of Chemical Engineering, Carnegie Mellon University, Pittsburgh, PA 15213, USA * Correspondence: [email protected]; Tel.: +1-412-268-7673 Received: 17 November 2020; Accepted: 9 December 2020; Published: 13 December 2020 Abstract: Recent efforts to determine the high-resolution crystal structures for the adenosine receptors (A1R and A2AR) have utilized modifications to the native receptors in order to facilitate receptor crystallization and structure determination. One common modification is a truncation of the unstructured C-terminus, which has been utilized for all the adenosine receptor crystal structures obtained to date. Ligand binding for this truncated receptor has been shown to be similar to full-length receptor for A2AR. However, the C-terminus has been identified as a location for protein-protein interactions that may be critical for the physiological function of these important drug targets. We show that variants with A2AR C-terminal truncations lacked cAMP-linked signaling compared to the full-length receptor constructs transfected into mammalian cells (HEK-293). In addition, we show that in a humanized yeast system, the absence of the full-length C-terminus affected downstream signaling using a yeast MAPK response-based fluorescence assay, though full-length receptors showed native-like G-protein coupling.
    [Show full text]
  • GNAI1 Antibody Cat
    GNAI1 Antibody Cat. No.: 26-905 GNAI1 Antibody Antibody used in WB on Human brain at 0.2-1 ug/ml. Antibody used in WB on Hum. Fetal Brain at 1 ug/ml. Specifications HOST SPECIES: Rabbit SPECIES REACTIVITY: Drosophila, Human, Mouse, Rat Antibody produced in rabbits immunized with a synthetic peptide corresponding a region IMMUNOGEN: of human GNAI1. TESTED APPLICATIONS: ELISA, WB GNAI1 antibody can be used for detection of GNAI1 by ELISA at 1:12500. GNAI1 antibody APPLICATIONS: can be used for detection of GNAI1 by western blot at 1 μg/mL, and HRP conjugated secondary antibody should be diluted 1:50,000 - 100,000. POSITIVE CONTROL: 1) Cat. No. XBL-10123 - Fetal Brain Tissue Lysate PREDICTED MOLECULAR 40 kDa WEIGHT: September 27, 2021 1 https://www.prosci-inc.com/gnai1-antibody-26-905.html Properties PURIFICATION: Antibody is purified by peptide affinity chromatography method. CLONALITY: Polyclonal CONJUGATE: Unconjugated PHYSICAL STATE: Liquid Purified antibody supplied in 1x PBS buffer with 0.09% (w/v) sodium azide and 2% BUFFER: sucrose. CONCENTRATION: batch dependent For short periods of storage (days) store at 4˚C. For longer periods of storage, store STORAGE CONDITIONS: GNAI1 antibody at -20˚C. As with any antibody avoid repeat freeze-thaw cycles. Additional Info OFFICIAL SYMBOL: GNAI1 ALTERNATE NAMES: GNAI1, Gi ACCESSION NO.: NP_002060 PROTEIN GI NO.: 33946324 GENE ID: 2770 USER NOTE: Optimal dilutions for each application to be determined by the researcher. Background and References September 27, 2021 2 https://www.prosci-inc.com/gnai1-antibody-26-905.html Guanine nucleotide-binding proteins (G proteins) form a large family of signal-transducing molecules.
    [Show full text]
  • Supporting Information
    Supporting Information Jin et al. 10.1073/pnas.1418629112 SI Materials and Methods Santa Cruz Biotechnology, 1:100), anti–neural-specific β-tubulin − − − − Animals. Mice carrying the Gas1 (1), Shh (2), Cdo , Boc (3), (Tuj1, mouse, Millipore, 1:800), anti-Neurotrophin receptor P75 f − Smo (4), Gnaz (5), and transgenic Wnt1:Cre (6) alleles were (Rabbit, Millipore, 1:200), anti-GFP (rabbit, Invitrogen, 1:100), − − − previously described. For simplicity, Gas1 , Cdo ,andBoc were anti-mouse Gas1 (goat, R&D Systems, 1:200), and anti-HuC/D used for the Lac-Z knock-in allele of Gas1 and β-geo–internal (mouse, Molecular Probes, 1:100). Alexa Fluor 488- and Alexa ribosomal entry site (IRES) human placental alkaline phosphatase Fluor 568-conjugated secondary antibodies (Invitrogen) against (hPLAP) gene-trapped alleles of Cdo and Boc, respectively. Ap- specific species (goat, mouse, and rabbit) were used for detection propriate mating schemes were designed to generate embryos of (Molecular Probes, all at 1:1,000). DAPI (Sigma) was used at desired genotypes, including controls. Embryo stages are specified 1 μg/mL for staining of DNA. in the text. The vaginal plug date is designated as embryonic day 0.5 (E0.5), following convention. For genotyping, tail or embryo Neurosphere Culture. Neurosphere-like bodies were generated −/− sac DNAs were used. Oligonucleotide primers and conditions using whole guts dissected from E11.5 wild-type or Gas1 for PCR are described in corresponding publications and on the embryos as previously described (7–9). They were dissociated in f Jackson Laboratory (JAX) website. The Gas1 allele was gener- basal media by mechanical pipetting, then plated into culture ated for this work, and its characterization is detailed in Fig.
    [Show full text]
  • GNAT1 Gene G Protein Subunit Alpha Transducin 1
    GNAT1 gene G protein subunit alpha transducin 1 Normal Function The GNAT1 gene provides instructions for making a protein called alpha (a )-transducin. This protein is one part (the alpha subunit) of a protein complex called transducin. There are several versions of transducin made up of different subunits. Each version is found in a particular cell type in the light-sensitive tissue at the back of the eye (the retina), where it plays a role in transmitting visual signals from the eye to the brain. The transducin complex that contains a -transducin is found only in specialized light receptor cells in the retina called rods. Rods are responsible for vision in low-light conditions. When light enters the eye, a rod cell protein called rhodopsin is turned on ( activated), which then activates a -transducin. Once activated, a -transducin breaks away from the transducin complex in order to activate another protein called cGMP-PDE, which triggers a series of chemical reactions that create electrical signals. These signals are transmitted from rod cells to the brain, where they are interpreted as vision. Health Conditions Related to Genetic Changes Autosomal dominant congenital stationary night blindness At least two mutations in the GNAT1 gene have been found to cause autosomal dominant congenital stationary night blindness, which is characterized by the inability to see in low light. One of these mutations impairs the protein's ability to activate cGMP-PDE; the other mutation results in a protein that is constantly turned on (constitutively activated). Both of these mutations disrupt the pathway that creates visual signals to be sent from rod cells to the brain.
    [Show full text]
  • And Diabetes-Associated Pancreatic Cancer: a GWAS Data Analysis
    Published OnlineFirst October 17, 2013; DOI: 10.1158/1055-9965.EPI-13-0437-T Cancer Epidemiology, Research Article Biomarkers & Prevention Genes–Environment Interactions in Obesity- and Diabetes-Associated Pancreatic Cancer: A GWAS Data Analysis Hongwei Tang1, Peng Wei3, Eric J. Duell4, Harvey A. Risch5, Sara H. Olson6, H. Bas Bueno-de-Mesquita7, Steven Gallinger9, Elizabeth A. Holly10, Gloria M. Petersen11, Paige M. Bracci10, Robert R. McWilliams11, Mazda Jenab12, Elio Riboli16, Anne Tjønneland18, Marie Christine Boutron-Ruault13,14,15, Rudolf Kaaks19, Dimitrios Trichopoulos20,21,22, Salvatore Panico23, Malin Sund24, Petra H.M. Peeters8,16, Kay-Tee Khaw17, Christopher I. Amos2, and Donghui Li1 Abstract Background: Obesity and diabetes are potentially alterable risk factors for pancreatic cancer. Genetic factors that modify the associations of obesity and diabetes with pancreatic cancer have previously not been examined at the genome-wide level. Methods: Using genome-wide association studies (GWAS) genotype and risk factor data from the Pancreatic Cancer Case Control Consortium, we conducted a discovery study of 2,028 cases and 2,109 controls to examine gene–obesity and gene–diabetes interactions in relation to pancreatic cancer risk by using the likelihood-ratio test nested in logistic regression models and Ingenuity Pathway Analysis (IPA). Results: After adjusting for multiple comparisons, a significant interaction of the chemokine signaling À pathway with obesity (P ¼ 3.29 Â 10 6) and a near significant interaction of calcium signaling pathway with À diabetes (P ¼ 1.57 Â 10 4) in modifying the risk of pancreatic cancer were observed. These findings were supported by results from IPA analysis of the top genes with nominal interactions.
    [Show full text]