The X-ray crystal structure of the tyrosine kinase domain of the human insulin receptor has been determined by multiwavelength anomalous diffraction phasing and refined to 2.1 A resolution. The structure reveals the determinants of substrate preference for tyrosine rather than serine or threonine and a novel autoinhibition mechanism whereby one of the tyrosines that is autophosphorylated in response to insulin, Tyr 1,162, is bound in the active site.

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The formation of disulphide bonds is essential to the structure and function of proteins. These bonds rapidly form either cotranslationally or immediately post-translationally in the lumen of the endoplasmic reticulum. Native disulphide pairing for such proteins has been achieved in vitro; however, the rates of reassembly are slow and the conditions non-physiological. To account for these observations, Anfinsen et al. proposed that a 'disulphide interchange protein' was the in vivo catalyst of disulphide bond rearrangement. Other groups discovered an activity with similar characteristics that catalysed the reductive cleavage of insulin and may be associated with insulin degradation, although this result has been disputed. The enzyme involved, protein disulphide isomerase (PDI; EC 5.3.4.1), may be the in vivo catalyst of disulphide bond formation. Here we describe the sequence of cloned rat liver PDI complementary DNA which predicts a protein with two distinct regions homologous with Escherichia coli thioredoxin, a known cofactor in oxidation-reduction reactions. Each of these regions contains the presumed active site sequence Trp-Cys-Gly-His-Cys-Lys, suggesting that PDI, similar in action to thioredoxin, catalyses disulphide bond interchange via an internal disulphide-sulphydryl interchange. The cDNA predicts a signal peptide consistent with the view that PDI is a luminal endoplasmic reticulum protein. PDI messenger RNA, although ubiquitous, is more highly concentrated in secretory cells.

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Calcium-dependent protein kinase activities have been studied in nerve growth cone particles (GCPs) and compared with those of synaptosomes. GCPs contain a set of phosphoproteins qualitatively similar to that of synaptic nerve terminals. However, major quantitative differences appear to exist: whereas synapsin I phosphorylation is relatively weak, the major kinase substrates of GCPs are a 46,000-dalton membrane protein (calcium/calmodulin dependent) and two acidic proteins of 80,000 and 40,000 daltons, phosphorylated by a calcium/phospholipid-dependent protein kinase. The presence of synaptic kinase activities in GCPs is consistent with their neuronal origin. The role of these kinases in GCPs is not understood at present. They may be involved in growth-related functions and/or may prepare the sprouting neuron for synaptic function.

The deduced primary sequence of the cytoplasmic protein-tyrosine kinase domain of the insulin receptor contains a conserved kinase homology region (receptor residues 1002-1257) flanked by a juxtamembrane region and a C-terminal tail. A soluble 48-kDa derivative (residues 959-1355) containing these regions but lacking the first six residues of the juxtamembrane region had earlier been synthesized in Sf9 cells using a baculovirus expression system. The catalytic core of the kinase domain was studied first by proteolytic analysis of the 48-kDa kinase and then by expressing a series of truncated kinase domains in transiently transfected COS cells. Based on these studies, two core kinases of 34 (residues 985-1283) and 35 (residues 978-1283) kDa, respectively, were overexpressed in Sf9 cells. Biochemical characterization of the 35-kDa kinase revealed that the core kinase conserved the major functional properties of the native receptor kinase domain. Activity of the 35-kDa kinase toward a synthetic peptide increased more than 200-fold upon autophosphorylation, which occurred exclusively at Tyr-1158, Tyr-1162, and Tyr-1163; the largest increase was observed between bis- and trisphosphorylation of the kinase. The activated 35- and 48-kDa kinases were similar with respect to specific activity and ATP and Mg2+ requirements for peptide phosphorylation. Moreover, autophosphorylation appeared to initiate predominantly at Tyr-1162, immediately followed by phosphorylation at Tyr-1158 and then at Tyr-1163. The rate of autophosphorylation was dependent on enzyme concentration, consistent with a trans-phosphorylation mechanism. Finally, the 35-kDa kinase was crystallized, making possible elucidation of its three-dimensional structure by x-ray crystallography.

We have placed human insulin receptor cDNA into a vector under the control of the simian virus 40 (SV40) early promoter and tested its function by transient expression in microinjected Xenopus oocytes and by expression in stably transformed CHO cells. The precursor and the a and 13 subunits of the receptor were detected by immunoprecipitation from extracts of these cells. The human insulin receptor expressed in CHO cells specifically binds 12-labeled insulin but not insulin-like growth factor I, displays insulin-stimulated autophosphorylation of the 13 subunit, and mediates insulinstimulated 2-deoxyglucose uptake. We conclude that the human insulin receptor Is synthesized, processed normally, and functional in this heterologous cell system. Diabetes mellitus is caused either by a deficiency of insulin or by insensitivity of the target cells to insulin. This latter defect causes the majority of diabetes, and its incidence appears to be increasing (1). An understanding of the defect in these patients requires an understanding of the molecular mechanisms ofinsulin action. The manifold insulin responses in target cells are initiated by the binding of insulin to its receptor, an integral membrane glycoprotein composed of two a (Mr 135,000) and two 13 subunits (Mr 95,000) (2, 3). Insulin binding to the a subunit (4) of the receptor results in stimulation of intrinsic phosphokinase activity of the P3 subunit and autophosphorylation predominantly on tyrosine residues (5,6). How this activity is related to the unique aspects of the insulin response is unknown.Recently the human insulin receptor cDNA has been cloned by Ebina et al. (7) and by Ullrich et al. (8). The nucleotide sequence of the cDNA has revealed the primary structure of the single-chain insulin receptor precursor of 1382 amino acids (154 kDa). The a subunit (735 amino acids, 84 kDa) contains a cysteine-rich crosslinking domain; the 3-subunit (620 amino acids, 70 kDa) contains both the single transmembrane domain of the receptor and the tyrosine phosphokinase domain with the presumed ATP binding site and potential tyrosine phosphorylation sites (7,8). These features of the human insulin receptor and of the related epidermal growth factor receptor (9) provide the structural basis for understanding receptor function; they also intensify the mystery surrounding the specific biological effects associated with these molecules.To elucidate the mechanism(s) of insulin action, the elements of the insulin receptor must somehow be functionally isolated and reconstituted. A heterologous cell system in which the receptor is expressed and then assayed for functional reconstitution would provide an experimental system in which the biological response of the cell to insulin could be explored ultimately in molecular detail. In this paper we demonstrate that the human insulin receptor cDNA can be expressed in heterologous cell systems. By the use of a monoclonal antibody specific for the human insulin receptor (10), it is possible to discriminate between endogenous r...