Immunological detection of isoforms of the somatostatin receptor subtype, SSTR2.
Somatostatin (SRIF) induces its diverse physiological actions through interactions with different receptor subtypes. Multiple SRIF receptor subtypes have recently been cloned. To analyze the physical properties of receptor subtype SSTR2, two different peptide-directed antibodies were generated against SSTR2. Antibody "2e3," directed against the peptide SSCTINWPGESGAWYT (residues 191-206), corresponding to a region in the predicted third extracellular domain of mouse SSTR2, and antibody "2i4," directed against the peptide SGTEDGERSDS (residues 333-343) from the predicted cytoplasmic tail of mouse SSTR2, were developed. In Chinese hamster ovary (CHO) cells stably expressing the mouse SSTR2 gene (CHOB), the antibody 2e3 recognized specifically a protein of 93-kDa protein by immunoblotting. No specific immunoreactivity was detected by 2e3 in nontransfected CHO cells or CHO cells stably expressing vector alone or human SSTR1 or mouse SSTR3 genes. The antibody 2i4 specifically immunoprecipitated SSTR2 solubilized from CHOB cells that could be labeled with the SSTR2-specific ligand 125I-MK-678. Furthermore, both 2e3 and 2i4 specifically immunoprecipitated 93-kDa [35S]methionine-labeled proteins from CHOB cells, indicating that they recognize the same proteins. In contrast to studies in CHOB cells, immunoblotting studies showed that 2e3 detected specifically a single 148-kDa protein from different regions of the rat brain that have previously been shown to express high levels of SSTR2 mRNA and SRIF receptors with high affinity for 125I-MK-678. In contrast, no immunoreactivity was detected in rat kidney, liver, or lung, which do not express SSTR2. No 93-kDa protein was detected specifically in the rat brain.(ABSTRACT TRUNCATED AT 250 WORDS)
Theveniau
M A
MA
Department of Pharmacology, University of Pennsylvania, School of Medicine, Philadelphia 19104.
Yasuda
K
K
Bell
G I
GI
Reisine
T
T
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies; D001921:Brain / Q000378:metabolism; D016466:CHO Cells; D002462:Cell Membrane / Q000378:metabolism; D006224:Cricetinae; D000939:Epitopes / Q000032:analysis; D006801:Humans; D015151:Immunoblotting / Q000379:methods; D008715:Methionine / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010455:Peptides / Q000276:immunology; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000032:analysis / Q000096:biosynthesis; D051381:Rats; D017481:Receptors, Somatostatin / Q000032:analysis* / Q000096:biosynthesis / Q000276:immunology; D014162:Transfection; D014316:Tritium
D000906:Antibodies; D000939:Epitopes; D010455:Peptides; D012333:RNA, Messenger; D017481:Receptors, Somatostatin; D014316:Tritium; D008715:Methionine