Frank P Leu1, Minesh Nandi. 1. 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
Abstract
OBJECTIVES: 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.
OBJECTIVES: 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.