The insulin receptor (IR) is a heterotetrameric glycoprotein composed of two alpha and two beta subunits linked by disulfide bonds. The alpha subunit is extracellular and binds insulin, while the beta subunit is transmembranal and functions as a cytosolic tyrosine protein kinase. Both alpha and beta subunits are coded for by a single mRNA whose gene is located on Chromosome 19p. The overall objective of this proposal is to study how inherited variations in the alpha subunit of the human IR gene alter cell physiology. To accomplish this goal we have identified families whose probands express monogenic, severe, insulin resistance, hypoglycemia, and leprechaunism. When insulin binding to their cultured skin fibroblasts is used as a genetic discriminant, an autosomal recessive pattern of inheritance emerges in which probands' cells have specific absence of high-affinity, insulin binding and parents' cells have partial impairment. These observations imply mutations in the IR alpha subunits. Glucose transport by cells from all probands is insulin-insensitive but one patient's cells have markedly increased, basal glucose transport. We will test the hypothesis that different mutations in the IR alpha subunit are the cause for those phenotypes of insulin resistance and loss of glucose homeostasis. To this end we will evaluate the structure of normal and mutant IR using affinity crosslinking of 125I-insulin to alpha subunits, 32P-ATP incorporation into beta subunits, and immunoblotting of the intact and disassociated IR. We will compare cellular signaling using insulin-enhanced, IR autophosphorylation, exogenous protein phosphorylation, and regulation of membrane transport. To clarify the relationship between increased, insulin-insensitive glucose uptake and mutations in the IR alpha subunit, we will transfect this unique proband's fibroblasts with normal IR cDNA. When insulin binding is normalized, we will evaluate glucose transporter function synthesis, turnover and gene expression. Transmission of the IR gene in these families will be explored by RFLP analysis. Preliminary RFLP analyses identified rare variations in the IR alpha subunit DNA which were transmitted with Mendelian patterns but revealed no gross deletions or insertions. Similarly normal and mutant IR mRNAs had the same size and quantity. We will procede to identify presumptive base pair substitutions in the alpha gene using ribonuclease cleavage at RNA:DNA mismatches. The area of interest of the mutant cDNA will be cloned and sequenced. In this way we will develop molecular methods of predicting inherited insulin resistance and clarify a novel signal by which the IR alpha subunit may regulate glucose transporter activity.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK040362-03
Application #
3240575
Study Section
Metabolism Study Section (MET)
Project Start
1988-09-01
Project End
1991-12-31
Budget Start
1990-09-01
Budget End
1991-12-31
Support Year
3
Fiscal Year
1990
Total Cost
Indirect Cost
Name
Emory University
Department
Type
Schools of Medicine
DUNS #
042250712
City
Atlanta
State
GA
Country
United States
Zip Code
30322
Longo, N; Singh, R; Elsas, L J (2001) Decreased half-life of insulin-like growth factor I in Rabson-Mendenhall syndrome. J Inherit Metab Dis 24:546-50
Longo, N; Langley, S D; Still, M J et al. (1995) Prenatal analysis of the insulin receptor gene in a family with leprechaunism. Prenat Diagn 15:1070-4
Longo, N; Langley, S D; Griffin, L D et al. (1995) Two mutations in the insulin receptor gene of a patient with leprechaunism: application to prenatal diagnosis. J Clin Endocrinol Metab 80:1496-501
Longo, N; Singh, R; Griffin, L D et al. (1994) Impaired growth in Rabson-Mendenhall syndrome: lack of effect of growth hormone and insulin-like growth factor-I. J Clin Endocrinol Metab 79:799-805
Longo, N; Langley, S D; Griffin, L D et al. (1993) Activation of glucose transport by a natural mutation in the human insulin receptor. Proc Natl Acad Sci U S A 90:60-4
Longo, N; Griffin, L D; Langley, S D et al. (1992) Glucose transport by cultured human fibroblasts: regulation by phorbol esters and insulin. Biochim Biophys Acta 1104:24-30
Longo, N; Langley, S D; Griffin, L D et al. (1992) Reduced mRNA and a nonsense mutation in the insulin-receptor gene produce heritable severe insulin resistance. Am J Hum Genet 50:998-1007
Longo, N; Langley, S D; Griffin, L D et al. (1992) Mutations in the insulin receptor and their effect on glucose transport. Trans Assoc Am Physicians 105:204-13
Longo, N; Shuster, R C; Griffin, L D et al. (1992) Activation of insulin receptor signaling by a single amino acid substitution in the transmembrane domain. J Biol Chem 267:12416-9
Elsas, L J; Longo, N (1992) Glucose transporters. Annu Rev Med 43:377-93

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