Ghsr Gene Structure

Determination of the nucleotide sequence of clone 7-3 revealed that it encoded a GPC-R with seven transmembrane a helical domains (7-TM) (Fig. 4). However, the GHS-R gene was truncated at its amino terminus by 13 amino acids (16). Using clone 7-3 as a hybridization probe, additional GHS-R cDNA and genomic clones were obtained from swine, human, rat, and mouse cDNA and genomic DNA libraries (17).

Two types of GHS-R cDNAs were isolated. Type 1a encoded a 7-TM GPC-R with binding and functional properties expected of a receptor for GHSs. The deduced amino acid sequence of the GHS-R highlights features in common with other GPC-Rs, which include conserved cysteine residues in the first two extracellular loops and several potential sites for co/posttranslational modifications (N-linked glycosylation and phosphorylation) and most importantly, the GPC-R signature aromatic triplet sequence (E/DRY) found immediately after TM-3 in the second intracellular loop (18).

Type 1b GHS-R cDNA represents an inactive, C-terminally truncated GPC-R that encoded only five predicted TM domains. The deduced amino acid sequence of type 1a and 1b cDNAs was identical up to leucine-265 (the second amino acid of TM-6) with the type 1b cDNA nucleotide sequence diverging and extending for an additional 24 amino acids.

The type 1a and 1b cDNAs are derived from a single gene by alternative mRNA processing. A genomic clone encoding the human and mouse GHS-R gene was isolated and a partial nucleotide sequence determined (Fig. 5). The human GHS-R gene is divided into two exons by a single intron of ~2 kb in length. Determination of the nucleotide sequence for the proposed human exon-intron boundaries and the complete intron of the human gene confirmed that the intron divides the ORF into an amino-terminal segment ending at leucine-265 (encompassing the extracellular domain, TM-1 through TM-5, and the three intra- and first two extracellular loops) and a carboxyl-terminal segment encoding TM-6, the third extracellular loop, TM-7, and the C-terminal intracellular domain. The position of the intron is highly conserved among rat, human, and swine GHS-R genes. Type 1a cDNA encodes the complete 7-TM GHS-R and results from a splicing event that removes the intron. With type 1b cDNA, the intron is not removed and the reading frame extends into the intron. cDNA analysis indicates that an alternative poly (A)+ addition site is used, which is presumably located in the intron. As a result, the

Fig. 3. Identification of swine pituitary cRNA library pool S10-20 and fractionation leading to isolation of a single GHS-R cDNA clone. A GHS-R response could be identified in Xenopus oocytes injected with cRNA pool S10-20 (complexity 10,000 cRNAs; aequorin chemilumines-cence). This pool was broken down, resulting in pool 271, which contained 1000 individual cRNAs. Pool 541 (500 cRNAs) was subsequently broken down resulting in the isolation of clone 7-3 (aequorin luminescence, left; Ca2+ activate chloride current in Xenopus oocytes, right).

Fig. 3. Identification of swine pituitary cRNA library pool S10-20 and fractionation leading to isolation of a single GHS-R cDNA clone. A GHS-R response could be identified in Xenopus oocytes injected with cRNA pool S10-20 (complexity 10,000 cRNAs; aequorin chemilumines-cence). This pool was broken down, resulting in pool 271, which contained 1000 individual cRNAs. Pool 541 (500 cRNAs) was subsequently broken down resulting in the isolation of clone 7-3 (aequorin luminescence, left; Ca2+ activate chloride current in Xenopus oocytes, right).

Fig. 4. Deduced amino acid sequences of GHS-Rs from swine, human, and rat. Schematic representation of the human swine and rat GHS-R (see inset at top right corner) as a 7 TM GPC-R. Individual amino acid residues are shown (single letter amino acid code). Transmembrane domains are numbered. The inset at the bottom left reveals the overall amino acid identities of the GHS-Rs from human swine and rat.

Fig. 4. Deduced amino acid sequences of GHS-Rs from swine, human, and rat. Schematic representation of the human swine and rat GHS-R (see inset at top right corner) as a 7 TM GPC-R. Individual amino acid residues are shown (single letter amino acid code). Transmembrane domains are numbered. The inset at the bottom left reveals the overall amino acid identities of the GHS-Rs from human swine and rat.

Fig. 5. The human GHS-R gene: physical map and nucleotide sequence of its exon-intron boundaries. The open box represents the coding sequence of TM1-5, the shaded box the coding sequence of TM6 and 7. The single intron of the GHS-R is outlined by the thin line separating the coding exons. Sizes of the restriction enzyme fragments (in kb) are indicated above the physical map. The nucleotide sequence at the exon-intron (upper and lower case, respectively) boundaries is shown just below the physical map. The structure of the type 1a and type 1b cDNAs, which diverge at amino acid 265, is shown below the physical map.

Fig. 5. The human GHS-R gene: physical map and nucleotide sequence of its exon-intron boundaries. The open box represents the coding sequence of TM1-5, the shaded box the coding sequence of TM6 and 7. The single intron of the GHS-R is outlined by the thin line separating the coding exons. Sizes of the restriction enzyme fragments (in kb) are indicated above the physical map. The nucleotide sequence at the exon-intron (upper and lower case, respectively) boundaries is shown just below the physical map. The structure of the type 1a and type 1b cDNAs, which diverge at amino acid 265, is shown below the physical map.

human and swine type 1b cDNA contain a short, 24-amino acid open reading frame fused to leucine-265, which is conserved in humans and swine.

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