Richard W. Furlanetto

Cloning of human p55γ, a regulatory subunit of phosphatidylinositol 3-kinase, by a yeast two-hybrid library screen with the insulin-like growth factor-I receptor

We have used the yeast two-hybrid system to identify proteins that interact with the intracellular domain of the insulin-like growth factor-I receptor (IGFIR). In a search of a human fetal brain library we identified a cDNA encoding a protein that is the human homologue of mouse p55PIK, a regulatory subunit of phosphatidylinositol 3-kinase (hp55 gamma). The hp55 gamma protein interacts strongly with the activated IGFIR but not with the kinase-negative mutant receptor. hp55 gamma also interacts with the insulin receptor (IR) in the yeast two-hybrid system. The putative hp55 gamma protein is composed of a unique amino terminal region followed by a proline-rich motif and two Src homology 2 (SH2) domains, which are highly homologous to those in mouse p55PIK, rat p55 gamma, human p85 alpha and bovine p85 beta; it contains no SH3 domain. hp55 gamma mRNAs are expressed in most human fetal and adult tissues with particularly high abundance in adult testis. Splice variant(s) of hp55 gamma, one of which has a deletion of 36 amino acids at the amino terminus and another which has an insertion of 59 amino acids at position 256 between the SH2 domains, were also identified. A GST-hp55 gamma fusion protein interacts in vitro with both the activated IGFIR and IR derived from mammalian cells. Our findings suggest that hp55 gamma interacts with the IGFIR and IR and may be involved in PI 3-kinase activation by these receptors.

Comparison of Transdermal and Oral Estrogen Therapy in Girls with Turner's Syndrome

Eight girls with Turners syndrome were given low dose oral ethinyl estradiol or transdermal 17 beta-estradiol in order to compare the effect of the route of administration on selected markers of hepatic metabolism, and various hormonal concentrations. Oral estrogen was given at a dose of 100 ng/kg/day and transdermal estrogen via adhesive skin patch at 0.0125 mg/kg/day. The subjects received one form of estradiol for one month, and after a one month washout period, received the other form. Both oral and transdermal estradiol caused a significant decrease in FSH while only transdermal resulted in a significant decrease in LH. Oral estradiol, though not transdermal estradiol, increased serum high density lipoprotein, thyroxine binding protein and growth hormone binding protein. Urinary growth hormone excretion increased after both forms of therapy, while insulin-like growth factor-I and insulin-like growth factor binding protein-3 remained unchanged. Thus, in girls with Turners syndrome, estrogen replacement by the transdermal route may have less deleterious effect on hepatic metabolism than oral estrogen.

Homozygous nonsense mutation in the insulin receptor gene of a patient with severe congenital insulin resistance: leprechaunism and the role of the insulin‐like growth factor receptor

Severe congenital insulin resistance in the syndrome of leprechaunism is caused by mutations in the insulin receptor gene. We report a patient with leprechaunism who was homozygous for a mutation resulting in the absence of cell surface insulin receptors. To determine whether the receptor for Insulin‐like growth factor‐I (IGF‐I) is involved in the phenotype of leprechaunism, we studied the effect of insulin and of IGF‐I on cells from this patient.

Mechanisms subserving insulin action in the gonad: Evidence that insulin induces specific phosphorylation of its immunoprecipitable receptor on ovarian cells

To test the capacity of insulin to work through a classical insulin-receptor pathway in the ovary, cultured swine granulosa cells were treated with insulin and/or increasing concentrations of insulin-receptor antiserum. Insulin-receptor antiserum but not control serum significantly (greater than 85%) attenuated insulins stimulation of progesterone biosynthesis. Moreover, in broken-cell preparations, insulin but not desoctapeptide insulin or somatomedins induced specific phosphorylation of the 95,000-dalton, immunoprecipitated beta subunit of the insulin receptor on ovarian cells. These observations provide the first evidence for discrete biochemical actions of insulin at the level of the cell-membrane receptor for insulin in gonadal cells.