Generation of polyclonal antiserum against the growth hormone secretagogue receptor (GHS-R): evidence that the GHS-R exists in the hypothalamus, pituitary and stomach of rats.
Growth hormone (GH) secretagogues (GHSs), which stimulate GH secretion, are synthetic compounds that act through the GHS receptor (GHS-R) which has been recently cloned. We raised an antiserum in a rabbit against a synthetic peptide corresponding to amino acid residues 248-260 of the third intracellular loop of the rat GHS-R. A competitive immunoassay showed that the antiserum had a specific affinity for the target peptide. To confirm the specificity of the antiserum, the GHS-R cDNA was stably expressed in COS-7 cells. In Western blot analysis, the band was detected at 44 kDa in the extracts from COS-7 cells expressing GHS-R (COS-7/tf3-2) but not in those from wild-type COS-7 cells. Furthermore, while COS-7/tf3-2 cells were strongly immunostained for GHS-R, no GHS-R-like immunoreactivity was observed in wild-type COS-7 cells. Immunoreactive bands were also observed at approximately 46 kDa in the extracts from rat hypothalamus, pituitary and stomach by Western blot analysis. These studies are the first to show the existence of GHS-R protein in the stomach. The antiserum for the GHS-R is sensitive and specific, and it would be useful for clarifying the roles of GHS/ghrelin.
Shuto
Y
Y
Department of Medicine, Nippon Medical School, Tokyo, Japan. shuto@nms.ac.jp
Shibasaki
T
T
Wada
K
K
Parhar
I
I
Kamegai
J
J
Sugihara
H
H
Oikawa
S
S
Wakabayashi
I
I
D000818:Animals; D000918:Antibody Specificity; D015153:Blotting, Western; D019556:COS Cells / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D005753:Gastric Mucosa / Q000378:metabolism; D007031:Hypothalamus / Q000378:metabolism*; D007106:Immune Sera / Q000096:biosynthesis* / Q000276:immunology / Q000302:isolation & purification; D010446:Peptide Fragments / Q000276:immunology*; D010902:Pituitary Gland / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D011817:Rabbits; D051381:Rats; D011956:Receptors, Cell Surface / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D043562:Receptors, G-Protein-Coupled; D054440:Receptors, Ghrelin; D011994:Recombinant Proteins / Q000276:immunology; D013270:Stomach / Q000737:chemistry*; D014018:Tissue Distribution; D014162:Transfection
D007106:Immune Sera; D010446:Peptide Fragments; D011956:Receptors, Cell Surface; D043562:Receptors, G-Protein-Coupled; D054440:Receptors, Ghrelin; D011994:Recombinant Proteins