IDENTIFICATION OF BINDING-PROTEINS FOR PERTUSSIS TOXIN ON PANCREATIC BETA-CELL-DERIVED INSULIN-SECRETING CELLS

The ability of pertussis toxin (PT) to recognize and bind to surface p roteins on cells derived from pancreatic insulin-secreting beta cells and alpha cell-like glucagon-producing cells was investigated employin g HIT-T15 (beta cell-derived) and In-R1-G9 (alpha cell-like) cell line s. PT recognition of membrane binding proteins on HIT-T15 and In-R1-G9 cells was first assessed with immunofluorescence microscopy in tissue culture. Both cell lines were equally well recognized by PT. N-octylg lucoside extracts of whole cells and isolated membranes were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PA GE) and blotted onto nitrocellulose membranes. PT, the B-oligomer, or the isolated PT dimers S2-S4 and S3-S4 recognized distinct proteins in HIT-T15 and In-R1-G9 cells of about 220 kDa. Recognition by the siali c acid specific Sambucus nigrica lectin identified these proteins as s ialoglycoproteins. Incubation of the blotted membrane proteins with si alidase or pretreatment of PT with anti-PT polyclonal antibodies aboli shed the recognition and binding of these proteins by PT. To demonstra te that these glycoproteins are also able to transduce PT mediated eff ects and thus might serve as PT binding proteins, the stimulation of i nsulin secretion in HIT-T15 cells was assessed. As the secretion of in sulin in HIT-T15 cells increased about 30% upon interaction with PT it was concluded that these glycoproteins are indeed functional as PT re ceptors.