Progress 07/01/08 to 06/30/11
Outputs
OUTPUTS: Isolation of guanylate cyclase receptors by RT-PCR from Malpighian tubules proved extremely challenging, as these proteins are synthesized from very large (4 kb), low abundance transcripts. My graduate student Chong Tang has now cloned four receptors that are either full length or nearly full length, from tissues of Manduca sexta. Three of these are most abundant in Malpighian tubule. One, NPR1, has 1,103 residues. NPR5 has 1,045 residues, and is most similar to a receptor for the eclosion hormone, reported by Chang et al. (2009) in the Oriental fruit fly Bactrocera dorsalis. NPR7 was cloned from CNS and is another large receptor of 1,166 residues, but incomplete at the N-terminus. NPR8 was also cloned from CNS; 1317 residues have been cloned but it is also incomplete. Gene precursors for NPR1 and NPR5 have had linkers attached and transferred into pcDNA5, and expressed in HEK293 cells. NPR5 responds very strongly to Tenebrio-ADFb. This is the main original goal of this project! Moreover, we find this receptor is the ortholog of a receptor published in PNAS in 2009 (V. 106, pps. 13371-6), which claimed it to be the receptor for the fly eclosion hormone. This paper is not correct and we will be submitting a paper to PNAS describing our results and contrasting them with those of Chang et al. PARTICIPANTS: Chong Tang has conducted all the research on this project. He has worked with Dr. Kidd in trying to locate receptors using in situ hybridization, but to date this has been unsuccessful. TARGET AUDIENCES: Entomologists and insect physiologists throughout the world will be interested in these results. PROJECT MODIFICATIONS: We changed to using Manduca sexta instead of Tenebrio molitor as this species is far larger, and the transcripts for these receptors are available only in TINY amounts. Later we can locate a beetle homolog of this receptor in the existing Tribolium castaneum genome.
Impacts
Beetles which attack trees are exceptionally sensitive to desication, as they must derive all of their water from metabolism of their diet. This is especially true for boring beetles, which eat only the relatively dry wood of the tree. Examples of genera which bore into wood are the Scolytidae, Lyctidae, Bostrichidae, Anobiidae, Cerambycidae, and Buprestidae. These genera should be susceptible to insect control agents which act on the control of water balance. This receptor is crucial in down-regulating water excretion by the Malpighian tubules, and chemicals which activate this receptor could disrupt the fluid homeostasis in the beetles, killing them.
Publications
- No publications reported this period
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Progress 01/01/10 to 12/31/10
Outputs
OUTPUTS: The isolation of guanylate cyclase receptors by RT-PCR from Malpighian tubules has proven an extremely challenging project. These proteins are synthesized from very large, low abundance transcripts. My graduate student Chong Tang has now cloned four receptors that are either full length or nearly full length, from tissues of Manduca sexta. Three of these are most abundant in Malpighian tubule. One, NPR1, has 1,086 residues. NPR5 has 1,092 residues, and is most similar to a receptor for the eclosion hormone, reported by Chang et al. (2009) in the Oriental fruit fly Bactrocera dorsalis. NPR7 was cloned from CNS and is another large receptor of 1,021 residues, but likely incomplete at the N-terminus. NPR8 was also cloned from CNS; it has 991 residues and is nearly complete. Q-PCR shows that NPR8 is highly expressed in Malpighian tubules. Clustal W suggests NPR8 is most similar of these to the ANP receptor of mammals, the most heavily studied guanylate cyclase receptor. Therefore of the three receptors we have identified from Malpighian tubule, it appears most likely that NPR1 is the receptor which responds to Tenmo-ADFa. A gene precursor for NPR1 has had linkers attached to it to allow transferring it into pcDNA5, a vector which will allow recombinant expression in HEK293 cells, which have been very widely used for expression and functional analysis of insect G protein coupled receptors. Once expressed, we shall treat the cells expressing it with Tenmo ADFa and analyzed for elevation of cGMP. PARTICIPANTS: Chong Tang is a graduate student in the Biochemistry Department who has done all of the molecular cloning in these studies. He is a very gifted, hard working experimentalist and has learned a great deal in this project. TARGET AUDIENCES: Insect physiologists interested in control of water balance will be quite interested in these results, and probably forest entomologists as well. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
We do not yet have publishable results, but we are close to getting there. Rather than having to clone a single very large receptor, we have cloned four, three of which are expressed in the Malpigian tubule, the target tissue of the antidiuretic hormone Tenmo-ADFa. By end of the project I am confident we shall have identified the receptor and determined its EC50 value for Tenmo-ADFa Control of water balance is vital in insects, and understanding the signaling that causes tubules to decrease their fluid secretion using this peptide is very important.
Publications
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Progress 01/01/09 to 12/31/09
Outputs
OUTPUTS: The isolation of guanylate cyclase receptors by RT-PCR from Malpighian tubules has proven an extremely challenging project. These proteins are synthesized from very large, low abundance transcripts. Analysis of genomic data for Drosophila melanogaster and Tribolium castaneum showed such receptors span ~40 KB in the genome, and introns are much larger than normal introns in insects. We made a strategic change to use Malpighian tubules from Manduca sexta , which are much larger than Tenebrio molitor, the focus of this proposal, because it is much easier to dissect enough tissue from Manduca to get enough material to amplify these rare messages. We have now succeeded in getting what we think is a full length sequence for one receptor, 1,015 AA, MW 122,868 Da; and a nearly full length sequence for another, 969 AA, MW 108,190 Da. Alignment of the latter receptor with homologues found in the Tribolium and Drosophila genomes suggests the second sequence may be missing 100-150 AA. PARTICIPANTS: Chong Tang, a fourth year Biochemistry and Molecular Biology graduate student, did an excellent job of cloning two very large receptors in the current reporting period. TARGET AUDIENCES: The international scientific community, interested in peptide hormones and their signaling mechanisms, will be quite interested in the data from this project when it is completed. Excellent progress has been made in the last year. PROJECT MODIFICATIONS: We changed from using Tenebrio molitor as experimental animal, to using Manduca sexta.Tenebrio adults weigh about 0.15 gram, whereas Manduca larvae weigh up to 10 gram. It is much easier to dissect sufficient Malpighian tubules from Manduca to be able to isolate the large, rare transcripts which are translated into these receptor proteins. Both animals respond with a strong antidiuretic response to the Tenebrio antidiuretic peptide, so we anticipate the receptors will be very similar in the two species.
Impacts
We suspect the larger of these two sequences is likely the Manduca homologue of the Tenebrio ADF receptor which is the subject of this project; it contains 12 Cys residues in the extracellular ligand binding domain. The smaller protein has only 5 Cys residues in the extracellular ligand binding domain; this is quite similar to the 6 Cys in the vertebrate ANP receptor. It is likely, but speculative, that it is an insect homologue of the ANP receptor, which would be expected to exist in the Malpighian tubule. We will need to create a construct containing the gene for the larger receptor, with appropriate linkers attached to the 5' and 3' ends, to transfer it into vertebrate cells for expression, followed by functional analysis. If it responds to low levels of Tenebrio ADFa, then it is the receptor for this peptide. Because of the large size of this receptor gene, it may be necessary to create the construct by ligating several smaller PCR products together. Thus we still face some challenges before saying we are confident that this is actually the ADF receptor. The good news, but complicating news, is that we found not one receptor, but two. There are thus currently no publishable results.
Publications
- No publications reported this period
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Progress 10/01/08 to 12/31/08
Outputs
OUTPUTS: For this recently started project, we proposed to isolate the receptor of Tenebrio molitor antidiuretic factor A, an exceptionally potent peptide which inhibits the secretion of Malpighian tubules in this beetle, and also inhibits secretion of mosquito tubules. To clone this receptor we proposed using RT-PCR on mRNA isolated from beetle Malpighian tubules using primers to highly conserved regions of the guanylate cyclase domain of the receptor. To test our techniques, we decided to start using these primers with a pre-existing cDNA library from Manduca sexta, which we already had available. The primers intended to use with the beetle receptor should also work for Manduca sexta, and in principle should also be useful for isolation of an insect equivalent of the atrial natriuretic peptide. (There is evidence that this peptide exists in tissues of Bombyx mori and Stomoxys calcitrans). However, we were unsuccessful in isolating a bona fide clone of either receptor from the Manduca sexta cDNA library. Detailed examination of the number of clones represented in this library suggest that the library is not of sufficient quality for detection of rare transcripts. Therefore, recent work continues using dissected Malpighian tubules from Tenebrio molitor. PARTICIPANTS: Chong Tang, graduate student TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: As mentioned, we started with an existing cDNA library from Manduca sexta Malpighian tubules to test the molecular biology methods. This is a challenging project because receptors are rare transcripts, and using degenerate probes can be difficult. We found the cDNA library was not of sufficient quality to contain rare transcrips.
Impacts
Identification of this receptor and its expression in cultured cells containing reporter molecules would allow screening of small molecules which may either stimulate or block the action of the receptor. This approach is used for screening for drugs. Half of all drugs work on peptide receptors; to date no pesticides have been identified which work using this approach.
Publications
- No publications reported this period
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