DISCOVERY AND CHARACTERIZATION OF GUANYLATE CYCLASE RECEPTORS IN

DISCOVERY AND CHARACTERIZATION OF GUANYLATE CYCLASE RECEPTORS IN MANDUCA SEXTA Chong Tang David A. Schooley

BACKGROUND • Guanylate Cyclase Receptors • Insect water homeostasis • Peptides activating c. GMP elevation

GUANYLATE CYCLASE RECEPOR • Three different types of receptor

GPCR AND IONIC CHANNEL RECEPTOR • Ionic channel receptor(ex. nicotinic receptor )-The binding of ligand changes the conductance properties of the receptor channel, thereby causing depolarization and tissue activation • G-protein coupled receptor Ligand binding domain Transmembrane domain Intracellular domain (ex. DH receptor)

BIOCHEMISTRY OF GUANYLATE CYCLASE RECEPTOR • Signal peptide • ECD • ICD(PKLD, GCD)

GUANYLATE CYCLASE RECEPTOR • Classfication: GC-A (NPRA-ANP, BNP), GC-B (NPRB-CNP), NPRC, GC-D, E, F (orphan receptors) • Function: NPRA- acts to reduce the water, sodium and adipose loads on the circulatory system, thereby reducing blood pressure • NPRB-various cardiovascular diseases • NPRC-clearance receptor • GCC-uroguanylin receptor • Soluble guanylate cyclase and receptor guanylate

SOLUBLE GUANYLATE CYCLASE • s. GC activity is regulated by NO, GTP, ATP and allosteric activator. • There are isoforms in animals including α 1, α 2, β 1 or β 2 subunits. Manduca have a different β 3. • SDIVGFIA, DVYKVETIGD and MPRYCLFG are more conserved. Bold characters (Gly, Arg, & Cys) determine the substrate specificity

ANP WATER HOMEOSTASIS Water osmosis

INSECT WATER HOMEOSTASIS • The primary organs of salt and water balance for insects are generally Malpighian tubules, salivary glands, midgut and hindgut • Malpighian tubules blind-end tubes secrete KCl or Na. Cl rich urine (by active transport), so water and solutes follow by passive diffusion • Reabsorption in ileum and rectum

DIURETIC PEPTIDES • The corticotropin-releasing factor related diuretic hormone family has over 30 identified members • DH ligands for GPCR, their binding elevates c. AMP concentration • increase Na+ conductance across Na+/K+/2 Cl- cotransporter • Increase Na+/H+ antiporter with a higher concentration of

ANTIDIURETIC PEPTIDES • those that inhibit MT secretion and those that stimulate fluid reabsorption (neuroparsins, CTSH, ITP, ADF) • chloride transport stimulating hormone (CTSH)- stimulate chloride transport into the rectum via c. AMP

PEPTIDES ACTIVATING CGMP ELEVATION • ANP/BNP c. GMP regulated/dependent proteins: c. GMP-dependent protein kinase (PKG), c. GMP selective phosphodiesterase (PDE), and cyclic nucleotide-gated ion channels. • renin, aldosterone, and angiotensin stimulate sodium and water retention • EH ADF ITP CHH CAP 2 b

INSECT ECDYSIS INVOLVED HORMONE-EH • The synthesis and secretion of a new cuticle by the epidermis, the subsequent shedding of the old cuticle (ecdysis), and the process of eclosion. • the preecdysis sequence (serves to loosen attachment between the old and new cuticle) and the ecdysis behavior (consists of rhythmic anterior-moving peristaltic waves which culminates in shedding of the cuticle). The ecdysis behavior is controlled by EH and ETH together. • Blood-borne EH acts on the Inka cell to cause c. GMP production and PETH/ETH depletion in a positive feedback loop {Zitnan, 2012}.

POSITIVE FEEDBACK LOOP • Eclosion hormone (EH) and ecdysistriggering hormone (ETH) work together on a number of targets to cause eclosion • Epitracheal glands (Inka cell) contain ETH • EH act on Inka cells to induce the release of PETH and ETH by c. GMP elevation {Zitnan, 2003 #6198} • ETH then acts on its receptor (ETHRA) in VM neurons in the brain to elicit further EH release.

EH RECEPTOR-GCR IN INSECT • EH receptor from Bactrocera dorsalis, Bdm. GC-1, a guanylate cyclase receptor {Chang, 2009} • Similar to NPR 5 • Isoform of Bdm. GC-1 and NPR 5

INSECT ANTIDIURETIC FACTORS: (ADF) • Tenmo-ADFa, is exceptionally potent (EC 50 ~ 10 f. M) {Eigenheer, 2002}, and is also a potent antidiuretic in Aedes aegypti, whereas Tenmo. ADFb is much less potent in Tenebrio, with an EC 50 of 240 p. M (in the cricket Acheta domesticus diuretic action) • The likely mode of action is that a c. AMP-specific phosphodiesterase is activated by c. GMP {Quinlan, 1998}, leading to the rapid degradation of c. AMP that mediates diuretic hormone activity.

ION TRANSPORT PEPTIDE: ITP • Ion transport peptide (ITP) in locusts was identified as having antidiuretic activity in the ileum, stimulating Cl- transport 10 fold, Na+ transport ~2 fold, K+ permeability ~3 fold, and isoosmotic fluid absorption ~4 fold • Knocking out ITP cause the mortality in ecdysis stage • ITP is a 72 amino-acid peptide that has ~40% sequence identity with crustacean hyperglycemic hormone (CHH)

• Protein kinase A, after stimulation by c. AMP, activates the apical Cl- pump and opens K+ and Cl- channels in the apical and basolateral membranes. • c. AMP can not mimic the function of ITP

INSECT DIURETIC PEPTIDE: CAP 2 B • CAP 2 b is a diuretic in Drosophila melanogaster, elevating c. GMP in the Malpighian tubule through the intermediacy of nitric oxide • Action Model: Stimulate phospholipase C, producing IP 3, and causing the release of Ca+2 from internal stores that stimulate entry of external Ca+2, activate a cascade consisting of Ca+2 -calmodulin-dependent nitric oxide synthase and, subsequently, guanylate cyclase and the VATPase • Quinlan et al. {, 1997} showed CAP 2 b, and c. GMP, to have an antidiuretic effect in Rhodnius prolixus.

CRUSTACEAN HYPERGLYCEMIC HORMONE-CHH • A family of neuropeptides include crustacean hyperglycemic hormone (CHH), molt inhibiting hormone (MIH), and vitellogenesis inhibiting hormone (VIH). • Different CHH isoforms have multifunctional roles in decapods; they play a central role in carbohydrate metabolism, but one (molt inhibiting hormone, MIH) also exerts an effect on the molt and reproduction. • CHH has been reported to have osmoregulatory functions in several species of crustaceans • In the lobster, the crustacean hyperglycemic hormone (CHH) has been shown to act on target tissues to elevate c. GMP level via a r. GCase {Goy, 1990}. More recently, Lee et al. {, 2007} have shown CHH and MIH to act via c. GMP in the crab.

EXPERIMENT • Searching for guanylate cyclase receptor • Clone guanylate cyclase receptor • Characterize guanylate cyclase receptor

• Alignment of receptors homologous to Rattus norvegicus GC-A in different species. Rattus norvegicus GC-A (AAI 28743), Bactrocera dorsalis (ACQ 90240), Tribolium castaneum (XP_970405), Bombyx mori (BGIBMGA 006891 PA), Aedes aegypti (XP_001653226), Apis mellifera (XP_001120461), Drosophila mojavensis (XP_001999659).

SEARCHING FOR GUANYLATE CYCLASE DOMAIN • RNA preparation • Reverse transcription • Degenerate PCR to get guanylate cyclase domain • Clone to T-vector and sequence

RNA PREPARATION Composition Final concentration weight Guanidinium thiocyanate 0. 8 M 118. 16 g Ammonium thiocyanate 0. 4 M 76. 12 g Sodium acetate (p. H 5) 0. 1 M 33. 4 ml of 3 M stock Glycerol 5% 50 ml Phenol 38% 380 ml Adjust p. H to 5

REVERSE TRANSCRIPTION • NEB MMLV reverse transcriptase, Roche reverse transcriptase AMV, Roche Transcriptor, Clontech SMART reverse transcriptase, and Clontech SMARTscribe reverse transcriptase (data are not shown).

DIFFICULTIES IN DEGENERATE PCR • The DVYKVETI degenerate primer (GAYGTNTAYAARGTNGARACNAT) has 2 X 4 X 2 X 4=1024 • RACE PCR is much more difficult that degenerate PCR • Touch-down program • Hot-start Taq is better than Pfu • No results on that

DEGENERATE PCR • NPR 5: AETYG-sense -T/C • 5' GCI GAR ACI TAT/C GG 3' • NPR 5: GCD-antisense • 5' CAGCAGCAGGCGC 3'

3 RACE PCR

5 RACE PCR

PCR BUFFER WITH COCL 2

5+3 RACE

Manduca sexta Bombyx BGIBMGA 006891 -TA Tribolium NCBI Drosophila NCBI XP_971364 CG 42637 A-C NPR 5 1092 AA NPR 6 500 AA 006799 -TA XP_970405. 1 CG 10738 -PA 003981 -TA&3982 - XP_975601. 2 TA CG 3216 -PB NPR 7 1022 AA NPR 8 1197 AA None found XP_001809661. 1 CG 33114_PB SP Q 9 BPR 0 NP_001167546. 1 CG 31183 NPR 1 1102 AA NPR 2 soluble NPR 3 soluble

Num Mg. Cl 2 (25 GC ber m. M) solution 1 48 2 0 DMSO (100%) d. NTP (10 m. M) Buffer Water 40 40 20 80 with 1. 8 m. M Mg. Cl 2 212 260 3 4 5 64 64 0 40 80 0 20 80 with 1. 8 m. M Mg. Cl 2 196 156 300 6 7 8 9 48 64 48 0 0 0 160 20 80 with 1. 8 m. M Mg. Cl 2 252 236 92 140 10 64 160 20 80 76 Table 8. Optimized conditions for PCR. All the figures are based on µl per 200 µl total volume. 5 x GC Buffer: 2. 5 M MMNO (N-Methylmorpholine N-oxide) with 2. 5 M proline, or 2 M MMNO with 2 M proline. The final working concentration is 0. 4 - 0. 5 M.

NPR 5 THIRD FRAGMENT

NPR 5 MUTATION

MUTAION TECHNOLOGY

BRIDGE THE PIECES

QPCR

PRELIMINARY HORMONE DOSE-RESPONSE CURVE FOR NPR 5 • c. GMP competitive binding assay

DISCCUSION-SEQUENCE ANALYSIS • Sequence analysis by signal. P server • Recently, Manduca sexta genome is nearly completed.

DISCUSSION-ALTERNATIVE SPLICING ISOFORMS • NPR 5 has a mutation between the kinase domain and the guanylate cyclase domain • ACGCCCCTCCGACCCCCACGCTGCTGCGGAGA CTCGCCATACCGGACCCGACGCCTTACAGGTATGTGTCATCTGCAACACTATAGCCACTAC TAACCAGCTGT • TACCTACAATTTATAAAATTAAATACTCTGTAAA TAGTAGTGACTCAGCCGTTGAACAAAAAACAAATATTAAAATGTATTTTCAGACCTCC TCTGGACGCTCTCG

FUNCTION OF MUTATION • In the closely related silkworm, there is no intron in this region. Tribolium neither. • This stop codon may not have a biological role • function as a dominant negative regulator of the functional receptor. • the splicing form without guanylate cyclase activity may function as a clearance receptor • NPR 1 has 3 different isoforms in the 3 end and 2 different isoforms in the 5 end

TISSUE LOCALIZATION • The Q-PCR data indicate that all of these receptors are expressed in the tubule, and four in the trachea, ileum, and head, but at lower levels • NPR 5 is the Manduca orthologue of the Bactrocera EH receptor • Very recent results of characterizating the NPR 5 receptor show that it responds similarly to Schgr-ITP and Tenmo. ADFb (the Manduca orthologs of ITP and ADF) • NPR 5 and NPR 8 are moderately abundant in tracheae, which is the respiratory system in Manduca • NPR 8 is most similar to the rat ANP receptor

NPR 6 • Immune function? • Metabolism function?

FUTURE WORK • RNAi in Tribolium • Produce antibody to do immunohistory • More biological analysis

REFERENCE • More in thesis

THANK YOU • First, I want to give my biggest appreciation to Dr. Schooley • Dr. Cushman (half year), Dr. Misono (two years) • Dr. Shintani, Dr. Tittiger • Dr. Keef, Dr. Blomquist • Dr. Harper