Precursor and gene structure of a growth hormone-releasing hormone-like molecule and pituitary adenylate cyclase activating polypeptide from sockeye salmon brain




Parker, David B .

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Growth hormone-releasing hormone (GHRH) is a neuropeptide which stimulates the synthesis and release of growth hormone (GH) from the pituitary gland. The primary structure of this peptide has been identified in 7 mammalian species while the gene has been isolated from only rat and human. GHRH is a member of the glucagon superfamily which includes vasoactive intestinal peptide (VIP), glucagon, secretin, peptide histidine methionine (PHM), gastric inhibitory peptide (GIP) and a recently identified peptide, pituitary adenylate cyclase activating polypeptide (PACAP). The evolutionary relationships of this superfamily are not well understood because the gene structure of these molecules has only been identified in mammals. This thesis presents immunological evidence of a GHRH-like molecule, and identifies a GHRH/PACAP precursor and gene that encode two peptides, a GHRH-like molecule structurally related to PACAP-related peptide (PRP) and PACAP, from sockeye salmon brain. An antiserum directed against a topologically assembled epitope of human GHRH 1-44 (NH2) was produced and used to develop a radioimmunoassay for detection of immunoreactive GHRH in brain extracts of salmon, guinea pig, mouse and alligator. An immunoreactive GHRH from salmon brain extracts with a retention time on reverse phase high-performance liquid chromatography (HPLC) distinct from human GHRH was present. In alligator, the same antiserum also detected a GHRH-like molecule. During attempts to purify alligator GHRH, alligator brain neuropeptide Y (NPY) was identified. Alligator NPY is the first non-mammalian vertebrate to have 100% sequence identity to human NPY. The sequence identity between alligator and human NPY suggests that this sequence is the same as the ancestral amniote NPY. Molecular biological techniques were used for the structural identification of the salmon GHRH-like molecule and another peptide. The salmon GHRH/PACAP precursor contains 173 amino acids and has dibasic and monobasic processing sites for cleavage of a 45 amino acid GHRH-like peptide with a free acid C-terminus and a 38 amino acid PACAP with an amidated C-terminus. The salmon GHRH-like peptide has 40% amino acid sequence identity with the human GHRH and 56% identity with human PACAP-related peptide (PRP). Salmon PACAP-38 is highly conserved (89%) with only 4 amino acid substitutions compared with the human, ovine and rat PACAP-38 peptides. Nucleotide sequencing and use of the polymerase chain reaction show the exon/intron organization of the salmon GHRH/PACAP gene to be similar to the human PACAP gene. Unlike the mammalian PACAP genes, the salmon gene produces two precursor forms by post-transcriptional processing. One form is similar to the mammalian PACAP precursors, while the second form is shorter due to the excision of exon 4. This deletion results in the loss of the first 32 amino acids of the GHRH-like peptide from the precursor. The high sequence identity and structural organization between the GHRH(PRP)/PACAP and PHM(PHI)/VIP genes suggest a duplication event occurred in an ancestral gene after the divergence from the other glucagon superfamily members. GHRH in mammals may have arisen by gene duplication after the divergence of the tetrapods from the other vertebrate lines. Thus, GH in fish may be controlled by the two molecules, GHRH-like peptide and PACAP, located on a single GHRH/PACAP gene.



Growth hormone releasing factor, Sockeye salmon