High affinity insulin binding by soluble insulin receptor extracellular domain fused to a leucine zipper.

Insulin receptors (IRs) that are truncated at the end of the ectodomain form dimers that bind insulin with different characteristics to wild type receptors. These soluble IRs have lowered affinity for insulin compared with full-length IR, and exhibit linear Scatchard plots in contrast to the curvilinear plots obtained with full-length IR, IR truncated at the C-terminus of the transmembrane region and IR ectodomains fused to the self-associating constant domains from Fc or lambda immunoglobulins. In this report, we have fused the IR ectodomain to the 33 residue leucine zipper from the transcriptional activator GCN4 of Saccharomyces cerevisiae. This fusion protein binds insulin with high affinity in a manner comparable to native receptor. The respective dissociation constants were Kd1 8.2 X 10(-11) M and Kd2 1.6 x 10(-8) M for hIRedZip and Kd1 5.7 x 10(-11) M and Kd2 6.3 x 10(-9) M for membrane-anchored, native receptor.

Mutational analysis of the N-linked glycosylation sites of the human insulin receptor.

Site-directed mutagenesis has been used to remove 15 of the 18 potential N-linked glycosylation sites, in 16 combinations, from the human exon 11-minus receptor isoform. The three glycosylation sites not mutated were asparagine residues 25, 397 and 894, which are known to be important in receptor biosynthesis or function. The effects of these mutations on proreceptor processing into alpha and beta subunits, cell-surface expression, insulin binding and receptor autophosphorylation were assessed in Chinese hamster ovary cells. The double mutants 16+78, 16+111, 16+215, 16+255, 337+418, the triple mutants 295+337+418, 295+418+514, 337+418+514 and 730+743+881 and the quadruple mutants 606+730+743+881 and 671+730+743+881 seemed normal by all criteria examined. The triple mutant 16+215+255 showed only low levels of correctly processed receptor on the cell surface, this processed receptor being autophosphorylated in response to insulin. The quadruple mutant 624+730+743+881 showed normal processing and ligand binding but exhibited a constitutively active tyrosine kinase as judged by autophosphorylation. Three higher-order mutants were constructed, two of which, 16+337+418+730+743+881 (Delta6) and 16+295+337+418+730+743+881 (Delta7a), seemed normal. The third construct, 16+337+418+514+730+743+881 (Delta7b), was expressed at high levels on the cell surface, essentially as uncleaved proreceptor with only the small proportion of Delta7b that was correctly processed showing insulin-stimulated autophosphorylation. The mutations of Delta6 and Delta7a were incorporated into soluble ectodomains, which had affinities for insulin that were 4-fold that of wild-type ectodomain. The Delta6 ectodomain expressed in Lec8 cells was produced in quantity in a bioreactor for subsequent structural analysis.

Single-molecule imaging of human insulin receptor ectodomain and its Fab complexes.

The insulin receptor (IR) is a four-chain, transmembrane dimer held together by disulfide bonds. To gain information about the molecular envelope and the organization of its domains, single-molecule images of the IR ectodomain and its complexes with three Fabs have been analyzed by electron microscopy. The data indicate that the IR ectodomain resembles a U-shaped prism of approximate dimensions 90 x 80 x 120 A. The width of the cleft (assumed membrane-distal) between the two side arms is sufficient to accommodate ligand. Fab 83-7, which recognizes the cys-rich region of IR, bound halfway up one end of each side arm in a diametrically opposite manner, indicating a twofold axis of symmetry normal to the membrane surface. Fabs 83-14 and 18-44, which have been mapped respectively to the first fibronectin type III domain (residues 469-592) and residues 765-770 in the insert domain, bound near the base of the prism at opposite corners. These images, together with the data from the recently determined 3D structure of the first three domains of the insulin-like growth factor type I receptor, suggest that the IR dimer is organized into two layers with the L1/cys-rich/L2 domains occupying the upper (membrane distal) region of the U-shaped prism and the fibronectin type III domains and the insert domains located predominantly in the membrane-proximal region.

Crystal structure of the first three domains of the type-1 insulin-like growth factor receptor.

The type-1 insulin-like growth-factor receptor (IGF-1R) and insulin receptor (IR) are closely related members of the tyrosine-kinase receptor superfamily. IR is essential for glucose homeostasis, whereas IGF-1R is involved in both normal growth and development and malignant transformation. Homologues of these receptors are found in animals as simple as cnidarians. The epidermal growth-factor receptor (EGFR) family is closely related to the IR family and has significant sequence identity to the extracellular portion we describe here. We now present the structure of the first three domains of IGF-IR (L1-Cys-rich-L2) determined to 2.6 A resolution. The L domains each consist of a single-stranded right-handed beta-helix. The Cys-rich region is composed of eight disulphide-bonded modules, seven of which form a rod-shaped domain with modules associated in an unusual manner. The three domains surround a central space of sufficient size to accommodate a ligand molecule. Although the fragment (residues 1-462) does not bind ligand, many of the determinants responsible for hormone binding and ligand specificity map to this central site. This structure therefore shows how the IR subfamily might interact with their ligands.

The disulfide bonds in the C-terminal domains of the human insulin receptor ectodomain.

The human insulin receptor is a homodimer consisting of two monomers linked by disulfide bonds. Each monomer comprises an alpha-chain that is entirely extracellular and a beta-chain that spans the cell membrane. The alpha-chain has a total of 37 cysteine residues, most of which form intrachain disulfide bonds, whereas the beta-chain contains 10 cysteine residues, four of which are in the extracellular region. There are two classes of disulfide bonds in the insulin receptor, those that can be reduced under mild reducing conditions to give alpha-beta monomers (class I) and those that require stronger reducing conditions (class II). The number of class I disulfides is small and includes the alpha-alpha dimer bond Cys524. In this report we describe the use of cyanogen bromide and protease digestion of the exon 11 plus form of the receptor ectodomain to identify disulfide linkages between the beta-chain residues Cys798 and Cys807 and between the alpha-chain Cys647 and the beta-chain Cys872. The latter bond is the sole alpha-beta link in the molecule and implies a side-by-side alignment of the two fibronectin III domains of the receptor. Also presented is evidence for additional alpha-alpha dimer bond(s) involving at least one of the cysteine residues of the triplet at positions 682, 683, and 685. Evidence is also presented to show that Cys884 exists as a buried thiol in the soluble ectodomain.

The evolution of support for safety-net hospitals.

The federal government, mostly through the Medicare and Medicaid programs, has created and maintained a set of structural mechanisms to support uncompensated care and clinical education: disproportionate-share hospital payments and direct and indirect graduate medical education payments. This paper provides a history of how these traditional supports have evolved. We note that the need to reduce federal and state spending threatens the level of these payments, while changes in the health care delivery system highlight a range of design and technical inadequacies in the current support mechanisms.

Crystallization of the first three domains of the human insulin-like growth factor-1 receptor.

The insulin-like growth factor-1 receptor (IGF-1R) is a tyrosine kinase receptor of central importance in cell proliferation. A fragment (residues 1-462) comprising the L1-cysteine rich-L2 domains of the human IGF-1R ectodomain has been overexpressed in glycosylation-deficient Lec8 cells and has been affinity-purified via a c-myc tag followed by gel filtration. The fragment was recognized by two anti-IGF-1R monoclonal antibodies, 24-31 and 24-60, but showed no detectable binding of IGF-1 or IGF-2. Isocratic elution of IGF-1R/462 on anion-exchange chromatography reduced sample heterogeneity, permitting the production of crystals that diffracted to 2.6 A resolution with cell dimensions a = 77.0 A, b = 99.5 A, c = 120.1 A, and space group P2(1)2(1)2(1).

Purification and properties of insulin receptor ectodomain from large-scale mammalian cell culture.

Ectodomain of the exon 11+ form of the human insulin receptor (hIR) was expressed in the mammalian cell secretion vector pEE6.HCMV-GS, containing the glutamine synthetase gene. Following transfection of the hIR ectodomain gene into Chinese hamster ovary (CHO-K1) cells, clones were isolated by selecting for glutamine synthetase expression with methionine sulphoximine. The expression levels of ectodomain were subsequently increased by gene amplification. Production was scaled up using a 40-liter airlift fermenter in which the transfected CHO-K1 cells were cultured on microcarrier beads, initially in medium containing 10% fetal calf serum (FCS). By continuous perfusion of serum-free medium into the bioreactor, cell viability was maintained during reduction of FCS, which enabled soluble hIR ectodomain to be harvested for at least 22 days. Harvests were concentrated 20-fold by anion-exchange chromatography. Optimal recovery of ectodomain from early harvests containing large quantities of serum proteins was achieved by insulin-affinity chromatography, whereas in later harvests purification was achieved by multistep chromatography. Analysis of the purified hIR ectodomain showed that it had a molecular weight by sedimentation equilibrium analysis of 269,500. Amino-terminal amino acid sequence analysis showed that the ectodomain was correctly processed to alpha and beta chains and that glycosylation characteristics were similar to those of native hIR. The integrity of the ectodomain was demonstrated by the recognition of conformation-dependent anti-hIR antibodies and by its binding of insulin (Kd approximately 2 x 10(-9) M). These results demonstrate the successful production and purification of hIR ectodomain by processes amenable to scale-up and in a form appropriate for structure/function studies of the ligand-binding domain of the receptor.

Determination of dehydroalanine residues in proteins and peptides: an improved method.

Dehydroalanine residues in peptides and proteins react with 4-pyridoethanethiol in alkaline solution to form 4-pyridoethyl cysteine residues, which after acid hydrolysis, give the corresponding amino acid. An experimental protocol has been developed, and with simple dehydroalanine derivatives, conversions of 96-99% are achieved. Tests with peptides and proteins show that serine and cysteine/cystine residues do not interfere with this analytical procedure. A sample of wool keratin contained 57 mumol dehydroalanine/g.

Faculty practice plans: the organization and characteristics of academic medical practice.

The contemporary academic medical center is a complex organization providing medical and other professional health education, biomedical and behavioral research, and a comprehensive range of patient care services. This paper presents data from the Association of American Medical Colleges' 1989 survey of 125 member faculty practice plans. The survey data showed that 62% of the 74 responding plans were units or associations within the medical school corporate structure. Plans were organized along a broad continuum from the autonomous, departmental model with decentralized governance and management to the group model with centralized governance and management. The growth of managed care, increased competition, and a greater reliance by the medical school on clinical practice income as a financing source are causing the practice plan to expand beyond billing of professional fees. The survey data showed that 75% of the practice plans operated satellite centers, and 61% planned to build new ambulatory care facilities in order to expand and improve services to patients. The practice plans also have adapted to changes in third-party reimbursement and are establishing mechanisms to negotiate managed care contracts involving multiple clinical departments to increase referrals and maintain patient shares; 86% of the plans participate in at least one managed-care organization. The role of the practice plan will continue to evolve in response to the needs of the academic medical center for a cooperative and supportive environment in which to conduct its traditional missions of teaching, research, and patient care.

31P nuclear magnetic resonance (NMR) identification of sugar phosphates in isolated rat ovarian follicular granulosa cells and the effects of follicle-stimulating hormone.

Immature female rats (23-30 days old) were implanted subcutaneously with diethylstilbestrol (DES) in silastic capsules. After 48 h their ovaries were removed and the granulosa cells isolated (Foreman et al. (1984) Life Sci. 35, 1273-1279). The cells were incubated in Hepes balanced saline buffer with substrates with or without follicle-stimulating hormone (FSH). At the end of incubation perchloric acid extracts were made for 31P NMR spectroscopy. The resonances of fructose 1-phosphate, fructose 6-phosphate, glucose 1-phosphate, and ribose 5-phosphate were identified in the granulosa cell extracts. The relative intensities of fructose 6-phosphate to ribose 5-phosphate decreased after incubation with FSH in vitro. This suggests that FSH increases the activity of the pentose pathway within 1 h. Thus, FSH can acutely activate those metabolic pathways which provide nicotinamide-adenine dinucleotide phosphate (NADPH) to be used in steroid synthesis and cholesterol mobilization.

The provision and financing of medical care for AIDS patients in US public and private teaching hospitals.

The National Association of Public Hospitals and the Association of American Medical Colleges' Council of Teaching Hospitals conducted a detailed survey on hospital care to patients with acquired immunodeficiency syndrome (AIDS) in major US public and private teaching institutions in 1985. The 169 hospitals treating patients with AIDS that responded to the survey reported providing inpatient services to 5393 patients with AIDS. These patients accounted for 171,205 inpatient days and 8806 inpatient admissions, with an average length of stay of 19 days. The average costs and revenue for patients with AIDS per day were $635 and $482, respectively, with Medicaid representing the most frequent third-party payer. The average inpatient cost per patient per year was $20,320. Using Centers for Disease Control estimates of 18,720 patients diagnosed as having AIDS and alive during any part of 1985, we estimate that the total cost of inpatient care for patients with AIDS was $380 million for that year. We also found significant regional and ownership differences in source of payment for patients with AIDS and regional differences in revenues received for AIDS treatment. Results indicate that the costs of treating patients with AIDS will profoundly affect major public and private teaching institutions, but that public teaching hospitals in states with restrictive Medicaid programs will be most adversely affected.

KIE: The authors report the results of a survey by the National Association of Public Hospitals and the Association of American Medical Colleges' Council of Teaching Hospitals regarding the provision and financing of care for AIDS patients by 169 major U.S. public and private teaching hospitals. In 1985, the average costs and revenues per day reported were $635 and $482 respectively, with Medicaid the most frequent third-party payer. Private hospitals treated more insured and homosexual patients while public institutions cared for higher percentages of Medicaid and drug abuse patients. The authors conclude that the study strongly suggests that major teaching hospitals and large city public institutions bear a disproportionate burden of AIDS care. They recommend less reliance on the hospital as the primary or sole source of care and the development of more cost-effective community-based services.

Graduate medical education: financing at the crossroads.

Teaching hospitals are concerned about the new competitive environment because their costs are generally higher than those of nonteaching hospitals. Many of the higher costs of teaching hospitals derive from their educational programs, the nature of the patient diagnostic case mix; losses on charity care; and their role in the introduction of new and more effective methods for prevention, diagnosis, and treatment of illness. All of these functions are important to the missions of teaching hospitals, and all make teaching hospitals more expensive to operate than nonteaching hospitals. While a solution to the problem of financing graduate medical education will not ensure teaching hospital financial health, a solution to the financing of graduate medical education will provide a more equitable environment in which teaching hospitals can compete. The basic question to be answered in the price competitive environment is, "can the teaching hospital continue to attract patients at a competitive price and maintain financial support for its educational programs at current levels?" Teaching hospitals are a diverse group of highly complex institutions performing medical education and research services for the nation and providing both basic and tertiary patient care. The current emphasis on reexamining national policies in light of more limited public resources places teaching hospitals and their vital activities at significant risk if their special nature and role are not appreciated.

Changing hospital payments: implications for teaching hospitals.

Hospitals cannot continue to view themselves only as social institutions whose performance will be assessed on the good they do. Teaching hospitals, in particular, cannot view themselves simply as distinctive combinations of social and educational institutions. Under Medicare's prospective pricing system, the hospital's role as production system is enhanced, and all hospitals must learn to balance the new economic realities as they work with their medical staff to adapt to a changed future.