GPCR somatostatin receptor extracellular loop 2 is a key ectodomain for making subtype-selective antibodies with agonist-like activities in the pancreatic neuroendocrine tumor BON cell line.
OBJECTIVE
The extracellular loop 2 (ECL2) ectodomain of the G protein-coupled receptor class A is thought to function like an inactivation "lid." We created polyclonal somatostatin receptor ECL2 (anti-SSTR ECL2) antibodies to target this lid and to examine if these antibodies can selectively activate the SSTR.


METHODS
Western blots and live-cell immunofluorescence microscopy determined anti-SSTR ECL2 antibody receptor binding selectivity. 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium assay (MTS assay) and cell cycle assay (fluorescence-activated cell sorting) checked for antibody effect on antiproliferation. Nexin assay examined the antibody's ability to induce apoptosis. LANCE cAMP kit (Perkin Elmer) detected antibody-dependent cAMP decrease. Enzyme-linked immunosorbent assay measured antibody effect on suppressing serotonin secretion. Ligand-receptor binding interference assay with the fluorescent somatostatin (FAM-SST) was used to examine antibody interference to SST-SSTR binding.


RESULTS
Anti-SSTR ECL2 antibodies are SSTR subtype selective and agonist-like, and they suppress cell proliferation via cell cycle arrest and apoptosis. In addition, these antibodies decrease cAMP production and inhibit serotonin secretion. Interestingly, these antibodies do not interfere with SST-SSTR binding.


CONCLUSIONS
The ECL2 is an important ectodomain for G protein-coupled receptor activation and required for ligand binding selectivity. The anti-SSTR2, anti-SSTR3, and anti-SSTR5 ECL2 antibodies independently inhibited BON proliferation and decreased hormone secretion. Unlike octreotide, our antibodies do not interfere with SST-SSTR binding.
Department of Pediatrics, The Cancer Institute of New Jersey, Verto Institute LLC, Neuroendocrine Tumor Research, UMDNJ, New Brunswick, NJ 08901, USA. fleu@verto-institute.org
Frank P
FP
Leu
Minesh
M
Nandi
D000911:Antibodies, Monoclonal / Q000276:immunology* / Q000378:metabolism / Q000494:pharmacology; D000918:Antibody Specificity / Q000276:immunology; D017209:Apoptosis / Q000187:drug effects; D001665:Binding Sites / Q000235:genetics / Q000276:immunology; D015153:Blotting, Western; D002453:Cell Cycle / Q000187:drug effects; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000276:immunology* / Q000378:metabolism; D049109:Cell Proliferation / Q000187:drug effects; D000242:Cyclic AMP / Q000096:biosynthesis; D006801:Humans; D018358:Neuroendocrine Tumors / Q000276:immunology / Q000378:metabolism / Q000473:pathology; D010190:Pancreatic Neoplasms / Q000276:immunology / Q000378:metabolism / Q000473:pathology; D011485:Protein Binding / Q000276:immunology; D017481:Receptors, Somatostatin / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D011994:Recombinant Proteins / Q000276:immunology / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012701:Serotonin / Q000378:metabolism
D000911:Antibodies, Monoclonal; D017481:Receptors, Somatostatin; D011994:Recombinant Proteins; C103631:somatostatin receptor 3; C101130:somatostatin receptor type 1; D012701:Serotonin; C094573:somatostatin receptor 5; C093733:somatostatin receptor 2; D000242:Cyclic AMP