title: Single molecule imaging of supported planar lipid bilayer--reconstituted human insulin receptors by in situ scanning probe microscopy.
abstract: A 480-kDa disulfide-linked heterodimer single-pass transmembrane protein, the insulin receptor, is autophosphorylated upon insulin binding to its extracellular domain. Remarkably, the structural basis for this activation process remained largely unknown until the recent cryoelectron microscopy studies of the insulin-insulin receptor complex by Luo et al. [Science 285 (1999) 1077]. We report here the results of an in situ study by high-resolution scanning probe microscopy of the full-length insulin receptor reconstituted within supported planar lipid bilayers. Our preliminary studies confirm that (1) the intact receptor can be reconstituted constitutively within a lipid vesicle and (2) fusion of the receptor-containing vesicles to mica resulted in the formation of molecular flat 5.5-nm-thick supported planar bilayers populated by two populations of protrusions, the shape and size of which are consistent with those of the insulin receptor's intra- and extracellular domains as modeled by the cryo-EM data of Ottensmeyer et al. [Biochemistry 39 (2000) 12103]. These results establish a framework for real-time studies of insulin-insulin receptor binding by in situ SPM and single molecule force spectroscopy.
authors.affiliation: Department of Biochemistry, University of Toronto, 4 Taddle Creek Road, Toronto, Ont., Canada M5S 3G9.
authors.forename: Andrea
authors.initials: A
authors.lastname: Slade
authors.forename: Jeanne
authors.initials: J
authors.lastname: Luh
authors.forename: Sylvia
authors.initials: S
authors.lastname: Ho
authors.forename: Christopher M
authors.initials: CM
authors.lastname: Yip
mesh_terms: D000538:Aluminum Silicates; D006801:Humans; D008051:Lipid Bilayers / Q000737:chemistry; D008081:Liposomes; D018625:Microscopy, Atomic Force; D020527:Microscopy, Scanning Probe / Q000379:methods*; D008958:Models, Molecular; D017434:Protein Structure, Tertiary; D011972:Receptor, Insulin / Q000737:chemistry* / Q000378:metabolism / Q000648:ultrastructure; D013499:Surface Properties; D013997:Time Factors
chemical_list: D000538:Aluminum Silicates; D008051:Lipid Bilayers; D008081:Liposomes; D011972:Receptor, Insulin; C011934:mica