Characterization of an antibody to the human melatonin mt1 receptor.
Melatonin acts via high affinity, G-protein coupled, seven transmembrane domain receptors. To precisely localize these receptors, antibodies were raised in chickens against a 15 amino acid fragment at the intracellular C-terminal region of the human melatonin receptor subtype mt1 (DSSNDVADRVKWKPS, mt(1338-352)). A chimeric form of the receptor with a hydrophilic Flag peptide (DYKDDDDK) in sequence with the extracellular N-terminus (Flag-mt1) was generated by polymerase chain reaction and expressed in mammalian cell lines. An IgY antibody (Y31), which gave high antibody titres by enzyme-linked immunosorbent assay, was used to localize Flag-mt1 in stably transfected cells by immunofluoresence. Flag-mt1 localization with Y31 was identical to that obtained with the M5 antibody directed against the Flag epitope and was mainly localized to the Golgi apparatus with some staining at the cell surface. No staining was seen in untransfected cells with either antibody. Y31 staining was abolished using antibody preabsorbed with peptide antigen. Y31 immunofluorescence in fetal human kidney sections was restricted to nephrogenic regions and matched that of 2-((125)I)iodomelatonin binding and mt1 gene expression by in situ hybridization. Y31 was used to immunoprecipitate biotinylated membrane proteins from Flag-mt1 stably transfected and untransfected CHO cells. Western blotting of immunoprecipitated proteins revealed two major bands specific to stably transfected cells, one at 63 kDa and one at 86 kDa. The first band almost certainly corresponds to the glycosylated form of Flag-mt1 and the second band to receptor dimers. Thus, Y31 antibody is suitable for use in detecting the human mt1 receptor subtype in tissues and in transfected cells.
Williams
L M
LM
Molecular Physiology Group, The Rowett Research Institute, Aberdeen, UK. l.williams@rri.sari.ac.uk
Drew
J E
JE
Bunnett
N W
NW
Grady
E
E
Barrett
P
P
Abramovich
D R
DR
Morris
A
A
Slater
D
D
D000595:Amino Acid Sequence; D000818:Animals; D000918:Antibody Specificity; D016466:CHO Cells; D002645:Chickens; D006224:Cricetinae; D004530:Egg Yolk / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay; D004847:Epithelial Cells / Q000166:cytology; D005455:Fluorescent Antibody Technique; D006801:Humans; D007457:Iodine Radioisotopes; D007668:Kidney / Q000166:cytology; D008550:Melatonin / Q000378:metabolism / Q000494:pharmacology; D008969:Molecular Sequence Data; D011869:Radioligand Assay; D051381:Rats; D011956:Receptors, Cell Surface / Q000032:analysis* / Q000235:genetics / Q000276:immunology*; D018160:Receptors, Cytoplasmic and Nuclear / Q000032:analysis* / Q000235:genetics / Q000276:immunology*; D044094:Receptors, Melatonin; D014162:Transfection
D007457:Iodine Radioisotopes; D011956:Receptors, Cell Surface; D018160:Receptors, Cytoplasmic and Nuclear; D044094:Receptors, Melatonin; D008550:Melatonin