FUSION OF INSULIN-RECEPTOR ECTODOMAINS TO IMMUNOGLOBULIN CONSTANT DOMAINS REPRODUCES HIGH-AFFINITY INSULIN BINDING IN-VITRO

A unique feature of the insulin receptor is that it is dimeric in the absence of ligand, Dimerization of two adjacent transmembrane domain ( TMD) alpha helices has been shown to be critical in receptor kinase ac tivation, Moreover, previous work has suggested that the TMD is involv ed in stabilizing the high-affinity binding site; soluble receptors ex pressed after simple truncation at the ectodomain-TMD junction have re duced affinity for insulin. To further examine this issue, we have rep laced the TMD and intracellular domain of the soluble human insulin re ceptor (HIRs) with constant domains from immunoglobulin Fc and lambda subunits (HIRs-Fc and HIRs-lambda), Studies of receptor biosynthesis a nd binding characteristics were performed following transient transfec tion of receptor cDNAs into human embryonal kidney 293 cells. Each hyb rid receptor was initially synthesized as a single chain proreceptor, followed by cleavage into alpha- and beta-Fc or beta-lambda subunits. The majority of secreted protein appeared in the cell medium as fully processed heterotetramer, Fe fragments released from HIRs-Fc by papain digestion and analyzed by nonreducing SDS-polyacrylamide gel electrop horesis were dimeric, Furthermore, dissociation constants for both chi meras were similar to those for the full-length holoreceptor (wild-typ e receptor, K-d1 = 200 pM and K-d2 = 2 nM; HIRs-Fc, K-d1 = 200 pM and K-d2 = 40 nM; and HIRs-lambda, K-d1 = 200 pw and K-d2 = 5 nM). These r esults extend previous observations that dimerization of the membrane- proximal ectodomain is necessary to maintain an intact high-affinity i nsulin-binding site.