Insect Biochem. Molec. Biol. Vol. 27, No. 8/9, pp. 735–743, 1997  1997 Elsevier Science Ltd Pergamon PII: S0965-1748(97)00052-0 All rights reserved. Printed in Great Britain 0965-1748/97 $17.00 + 0.00 The Heliothis virescens 170 kDa Aminopeptidase Functions as “Receptor A” by Mediating Specific Bacillus thuringiensis Cry1A -Endotoxin Binding and Pore Formation KE LUO,² SREEDHARA SANGADALA,² LUKE MASSON,§ ALBERTO MAZZA,§ ROLAND BROUSSEAU,§ MICHAEL J. ADANG‡²* Received 6 March 1997; revised and accepted 18 July 1997 The relationship between Bacillus thuringiensis Cry1Aa, Cry1Ab and Cry1Ac -endotoxin binding and pore formation was investigated using a purified 170 kDa aminopeptidase N (APN) from Heliothis virescens brush border membranes. Aminopeptidases with molecular sizes of 110, 140 and 170 kDa were eluted from a Cry1Ac toxin affinity column using N- acetylgalactosamine. The 140 kDa aminopeptidase has a cross-reacting determinant typical of a cleaved glycosyl-phosphatidylinositol anchor. After mild base treatment to de-acylate the glycosyl-phosphatidylinositol linkage and incubation in phosphatidyl inositol phospholipase C, anti-cross-reacting determinant antibody recognized the 170 kDa protein. Kinetic binding characteristics of Cry1A toxins to purified 170 kDa APN were determined using surface plas- mon resonance. Cry1Aa, Cry1Ab and Cry1Ac, but not Cry1C and Cry1E toxins recognized 170 kDa APN. Each Cry1A toxin recognized two binding sites: a high affinity site with K D ranging from 41 to 95 nM and a lower affinity site with K D in the 325 to 623 nM range. N- acetylgalactosamine inhibited Cry1Ac but not Cry1Aa and Cry1Ab binding to 170 kDa APN. When reconstituted into phospholipid vesicles, the 170 kDa APN promoted toxin-induced 86 Rb + release for Cry1A toxins, but not Cry1C toxin. Furthermore Cry1Ac, the Cry protein most toxic to H. virescens larvae, caused 86 Rb + release at lower concentrations, and to a greater extent than Cry1Aa and Cry1Ab toxins. The correlation between toxin-binding specificity and 86 Rb + release strongly suggests that the purified 170 kDa APN is the functional receptor A in the H. virescens midgut epithelial cell brush border membranes.  1997 Elsevier Science Ltd. All rights reserved Bacillus thuringiensis Cry1A -endotoxins Heliothis virescens Aminopeptidase N Receptor GPI anchor Surface plasmon resonance Pore formation INTRODUCTION Bacillus thuringiensis Cry1 -endotoxins bind with high affinity to receptors on the brush border membrane of the midgut in susceptible insects. Toxin inserts into the *Author for correspondence. Tel: + 1 706 542 2436; Fax: + 1 706 542 2640; e-mail: Adang@bscr.uga.edu. ²Department of Entomology, University of Georgia, Athens, GA 30602-2603, U.S.A. ‡Department of Biochemistry and Molecular Biology, University of Georgia, Athens, GA 30602-2603, U.S.A. §Biotechnology Research Institute, National Research Council of Can- ada, 6100 Royalmount Avenue, Montreal, Quebec, Canada H4P2R2 735 epithelial cell membrane creating ion channels, causing cell lysis and insect mortality (Gill et al., 1992; Knowles and Dow, 1993). A three-site model fits the specificity of Cry1Aa, Cry1Ab, and Cry1Ac binding to receptors in Heliothis virescens brush border membranes (Van Rie et al., 1989). Receptor A binds Cry1Aa, Cry1Ab and Cry1Ac toxins. Receptor B binds Cry1Ab and Cry1Ac toxins but not Cry1Aa, and Receptor C binds only Cry1Ac toxin. Recent data suggests Receptor A is modified in a strain of B. thuringiensis-resistant H. virescens. Lee et al. (1995) reported an absence of Cry1Aa binding sites, but no decrease in Cry1Ab and Cry1Ac binding sites in Cry1Ac-resistant H. virescens. The altered binding site