Expression cloning of an insect diuretic hormone receptor. A member of the calcitonin/secretin receptor family.

Insect diuretic hormones and their receptors regulate fluid and ion secretion and thus are attractive targets for the design of novel insect control agents. A complementary DNA clone encoding a corticotropin-releasing factor-related diuretic hormone receptor from the tobacco hornworm Manduca sexta was isolated by expression cloning in COS-7 cells. The receptor consists of 395 amino acids and contains seven putative transmembrane domains. The expressed receptor binds M. sexta diuretic hormone, as well as several related insect diuretic peptides with high affinity. Furthermore, each of these peptides stimulate adenylate cyclase in COS-7 cells transfected with the receptor. The M. sexta diuretic hormone receptor is homologous to the receptors for calcitonin, secretin, vasoactive intestinal peptide, parathyroid hormone, glucagon-like peptide 1, growth hormone-releasing hormone, pituitary adenylate cyclase-activating polypeptide, and glucagon. The M. sexta diuretic hormone receptor is the first nonmammalian member of this family to be identified.

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to the GenBankTMIEMBL Data Bank with accession numberfs) U03489.
While considerable effort has been directed toward identifying insect diuretic hormones and their modes of action, a report characterizing an insect diuretic hormone receptor has appeared only recently (8). Mt membranes from M. sexta larvae were found to contain a receptor that bound 3H-labeled Mas-DH with extraordinarily high affinity (& = 79 PM). I have used an expression cloning strategy in COS-7 cells to isolate a cDNA that encodes a M. sexta  cDNA Library Construction--Total RNA was isolated from the Mts of 5th instar M. sexta larvae by homogenization in guanidinium isothiocyanate and centrifugation through cesium chloride. Polyadenylated RNA was selected by chromatography using oligo(dT)-cellulose, and directional cDNA was prepared using a Stratagene ZAP-cDNA synthesis kit according to the manufacturer's instructions (Stratagene, La Jolla, CA). The cDNA was size-selected on Sepharose CL-4B, and fractions containing cDNA 1 kb and larger were ligated into the EcoRI and XhoI sites of the mammalian expression vector pKSl (9). The library was electroporated into Escherichia coli PLK-F' and separated into 512 pools of approximately 2100 transformants each. Plasmid DNA was prepared from each pool by the alkaline lysis method (10). COS-7 Cell Dansfection and Library Screening-COS-7 cells were plasmid DNA by the DEAE-dextran method (10). After 3 days, the cells grown in six-well tissue culture plates and transfected with 1-2 pg of were screened for diuretic hormone binding by incubation with 80-100 PM Bolton-Hunter 12SI-labeled Mas-DH (8). After incubation in binding buffer (25 rn potassium phosphate (pH 6 3 , 240 rn sucrose, 0.2% bovine serum albumin) for 1 h a t 4 "C, the cells were washed three times with ice-cold binding buffer, fixed in phosphate-buffered saline containing 25 rn HEPES (pH 7) and 2% glutaraldehyde, dried, and coated with NTB-2 photographic emulsion (IBI, New Haven, CT). Plates were exposed for 3 days and developed. Binding of 3H-Labeled Mas-DH to Membranes of COS-7 Cells-COS-7 cells were grown in 150-cm2 tissue culture flasks and transfected with 15 pg of pKSliMas-DH-R by the DEAE-dextran technique and harvested after 3 days by treatment with 1 rn EDTA in phosphatebuffered saline. Cells were homogenized by sonication on ice and centrifuged at 1000 x g. The supernatant was then centrifuged at 107,000 x g, and the pellet was resuspended in 25 r m potassium phosphate (pH 6.5), 240 m~ sucrose, and 1 rn EDTA. The membranes were stored at -70 "C. Binding experiments were carried out as previously described (8) using 10 pg of COS-7 membranedassay tube in binding buffer.
cAMPAssay-COS-7 cells were grown in 25-cm2 tissue culture flasks and transfected with 3 pg of PKSliMas-DH-R by the DEAE-dextran technique. After 3 days the cells were incubated in cell media (Dulbec-CO'S modified Eagle's media and 10% fetal calf serum) containing 1 rn isobutylmethylxanthine and various concentrations of peptide hormones for 15 min at 37 "C. The reaction was terminated by aspiration of the cell media and addition of 7.5% trichloroacetic acid. CAMP was measured by radioimmunoassay ( h e r s h a m Corp.).
Northern Hybridization and Nucleotide Sequence Annlysis-One microgram of polyadenylated RNA from various tissues was separated on a 1.5% formaldehyddagarose gel, transfemed to nitrocellulose, and probed with a random primed, 32P-labeled, full-length cDNA of Mas-DH-R using standard procedures (10). The final wash was conducted in 0.1 x SSPE, 0.1% SDS at 60 "C for 15 min. The cDNA encoding the Mas-DH-R was sequenced in both directions by the dideoxy nucleotide chain termination method using Sequenase version 2.0 (U. S. Biochemical Corp.).

RESULTS
A cDNA library was constructed in the mammalian expression vector pKSl (9)  each. Miniprep plasmid DNA from each pool was transfected into COS-7 cells and screened by lz5I-1abeled Mas-DH photoemulsion autoradiography. Six pools out of 150 were found to specifically bind lZ51-labeled Mas-DH. The positive pool with the greatest number of labeled cells was subdivided until a single clone (pKSUMas-DH-R) was obtained. Fig. 1 shows a saturation isotherm of 3H-labeled Mas-DH binding to membranes of COS-7 cells transfected with pKSl/Mas-DH-R. Analysis of the binding data by LIGAND (11) indicated a single specific and saturable binding site displaying an apparent & of 56 PM. This value agrees closely with the Kd of Mas-DH receptors prepared from Mt membranes (8).
The clone pKSl/Mas-DH-R contains an insert of 1.6 kb that encodes a protein of 395 amino acids based upon the longest open reading frame (Fig. 2). An in-frame stop codon (nucleotides 143-145) upstream of the proposed initiator AUG codon (nucleotides 248-250) suggests that pKSl/Mas-DH-R contains the entire coding region of the Mas-DH receptor. Hydrophobicity analysis of the amino acid sequence by the method of Kyte and Doolittle (12) suggests the protein spans the membrane seven times. The Mas-DH receptor is the first insect-specific neuropeptide receptor to be cloned and is homologous to a recently discovered family of G-protein-coupled receptors first

receptor (rSec-R), porcine calcitonin receptor (pCT-R), oposeum parathyroid hormone receptor (okPTH-R), rat glucagon-like
Highlighted areas denote amino acids conserved between Mas-DH-R and other receptors. Putative transmembrane domains are indicated by lines above the Mas-DH receptor sequence. Asterisks indicate conserved cysteine residues and potential N-glycosylation sites in Mas-DH-R are marked by #. Alignment was performed using the Pileup program in the Genetics Computer Group software package (23). Each member of the caicitonid~retin receptor family that has been identified activates adenylate cyclase; it has been suggested that the amino acids comprising the third intracellular loop are involved in coupling to G-proteins in a manner analogous to the &-adrenergic receptor (22).

RG. 3. Displacement of sH-labeled Mas-DH
To determine the specificity of ligand binding to the recombinant receptor, several peptides related to Mas-DH were tested in competition with 3H-labeled Mas-DH for binding to the expressed receptor. The known CRF-related diuretic peptides displayed an order of affinity of Mas-DH = Pea-DP = Acd-DH > Lom-DH --Mas-DPII (Fig. 3). The apparent ICso values for Mas-DH, Pea-DP, and Acd-DH were -1 m, while the values for Lorn-DH and Mas-DPII were -8 and -12 m, respectively. Neither vasoactive intestinal peptide, growth hormone-releasing hormone, glucagon, nor p a r a t h~i d hormone inhibited binding of 3H-labeled Mas-DH to the recombinant Mas-DH receptor at concentrations as high as 1 (data not shown), Tb determine if the expressed receptor could stimulate adenylate cyclase, COS-7 cells transfected with pKSliMas-DH-R were incubated with Mas-DH or related peptides (Fig. 4). All five CRF-related diuretic peptides stimulated CAMP synthesis approximately 22-fold above untreated cells and the order of potency matched the order of a n i t y . Treatment of COS-7 cells expressing the rat bradykinin receptor with 10 ~l i Mas-DH did not stimulate CAMP synthesis. The median effective concentration (EC50) for s t~u l a t i o n of CAMP by Mas-DH in COS-7 cells domesticus. Thus, although Acd-DH is a potent Mas-DH receptor agonist, the Acd-DH receptor does not appear to have an overall high sequence identity with the Mas-DH receptor.

ISC CUSS ION
The results presented above indicate that I have cloned a diuretic hormone receptor from the insect M. sextu. The receptor has very high affinity for Mas-DH, is expressed in Mts but not in fat body or brain, and is coupled to a CAMP second messenger system. The receptor possesses seven putative transmembrane domains that are common to other G-pmteincoupled receptors. The Mas-DH-R is the first insect (and non-mammalian^ receptor identified that is a member of the calcitonidsecretin receptor family. This suggests that, from an evolution standpoint, this family is quite old. It is surprising that the other four known CRF-related diuretic peptides are potent agonists of the Mas-DN receptor, especially when one considers that the sequence identity of these peptides to Mas-DH is only in the range of 3450%. Hence, the receptor binding domains of these peptides should share common structural elements. The secondary structure of Mas-DH has been investigated by circular dichroism studies (8). The peptide appears to exist in a random form in an aqueous environment. However, in a hydrophobic environment, mimicked by trifluoroethanof, Mas-DH undergoes a transition to a helical conformation. It is possible that in vivo the peptide transforms to a helical con~ormation upon binding to the receptor. In this model, the helical domain of the peptide is positioned in the membrane and surrounded by transmembrane regions of the receptor. Computational analysis of the other known CRF-related diuretic peptides reveal that they also contain long helical region^.^ However, one must await experimental studies of the secondary structure of these peptides to confirm this finding. It is interesting to note that secretin, glucagon, growth hormonereleasing hormone, pituitary adenylate cyclase-activating polypeptide and vasoactive intestinal peptide also assume an ex- tended helical conformation in a hydrophobic environment (24). Hence, this common structural element may be important for receptorAigand interactions for members of the calcitonin/ secretin receptor family.
The cloning of the Mas-DH receptor will facilitate our understanding the mechanism of action of insect diuretic hormones. Furthermore, since the receptor is functionally coupled to a CAMP second messenger system in COS-7 cells, one can utilize this system to study both binding to and activation of the receptor by various analogs of Mas-DH. While receptors for the other known CRF-related diuretic peptides have not been characterized, they are likely to be homologous to the Mas-DH receptor since their ligands are related to Mas-DH and are strong Mas-DH receptor agonists. Hence, use of oligonucleotides derived from conserved regions in the calcitoninlsecretin family should facilitate the cloning of other CRF-related insect diuretic hormone receptors.