EFFECTS OF MIDGUT-PROTEIN-PREPARATIVE AND LIGAND-BINDING PROCEDURES ON THE TOXIN BINDING CHARACTERISTICS OF BT-R-1, A COMMON HIGH-AFFINITY RECEPTOR IN MANDUCA-SEXTA FOR CRY1A BACILLUS-THURINGIENSIS TOXINS

The identity of the physiologically important Cry1A receptor protein(s ) in the lepidopteran Manduca sexta has been a matter of dispute due t o the multiple proteins which bind the Cry1Ac toxin. Cry1Aa, Cry1Ab, a nd Cry1Ac exhibit essentially identical toxicities toward M. sexta lar vae and show a high degree of sequence and presumed structural identit ies. These similarities make it likely that there is a common mechanis m of toxicity in these lepidopteran-specific toxins in terms of both m ode of action and the receptor proteins through which these toxins exe rt their lepidopteran-specific toxicity. Investigators in our laborato ry previously demonstrated that the cloned 210-kDa glycoprotein BT-R-1 binds all three Cry1A toxins (T. P. Keeton and L. A. Bulla, Jr., Appl . Environ. Microbiol. 63:3419-3425, 1997). This protein remains a comm on binding protein evens after being subjected to various midgut membr ane preparation and processing protocols. The method used to isolate p roteins from the M. sexta larval midgut in no significant was affects the results of ligand binding and vacuum blotting experiments, and we have been unable to detect specific, high-affinity binding of any Cry1 A toxin to Cry1Ac binding proteins other than BT-R-1. Alterations in b lot substrate and blocking, hybridization, and washing buffers support these conclusions. Collectively, these results indicate that in M. se xta the cadherin-like BT-R-1 protein is a common high-affinity recepto r protein for the Cry1A family of toxins.