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Larissa Balogh Endocrine Laboratory California State University at Long Beach

The Growth Inhibitory Role and Cell Membrane Association of a Reptilian Insulin-Like Growth Factor Binding Protein. Larissa Balogh Endocrine Laboratory California State University at Long Beach Research Advisor: Dr. Kevin M. Kelley. Insulin-like growth factors.

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Larissa Balogh Endocrine Laboratory California State University at Long Beach

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  1. The Growth Inhibitory Role and Cell Membrane Association of a Reptilian Insulin-Like Growth Factor Binding Protein Larissa Balogh Endocrine Laboratory California State University at Long Beach Research Advisor: Dr. Kevin M. Kelley

  2. Insulin-like growth factors • Insulin-like growth factors (IGFs) are growth-stimulatory peptides • IGF-I receptor activates cell growth • Insulin receptor transduces metabolic functions

  3. INS/IGF Precursor (before vertebrate emergence) IGFBP introduced (early vertebrates?) INS IGF X ? IGFBP Cell Membrane INS-Receptor IGF-Receptor (metabolic) (growth) Slide by Dr. Kevin Kelley

  4. IGF-binding proteins • IGF-binding proteins (IGFBPs) are the integrators of the endocrine growth-regulatory apparatus • block IGF interaction with insulin receptors • regulate IGF bioactivity • IGF-independent effects

  5. Six principal mammalian IGFBPs • Little is known about non-mammalian IGFBPs • IgH-2 cell line • derived from heart epithelial cells • of the reptile, Iguana iguana

  6. IgH-2 Iguana heart cell • single IGFBP, binds 125I-IGF-I • binds cell membrane surface • removal of cell-surface IGFBP Photo by Dr. Underwood, CSULB using a Nikon E800 microscope with a magnifier CCD camera

  7. Hypothesis • The membrane-bound Iguana IGFBP serves an IGF-inhibitory role in the heart cells IGF arriving at the cellular site is removed from proximity to the growth receptor

  8. cell surface IGFBP IGF-I receptor

  9. Goals • Determine the effects of IGF-I on cell-surface associated IGFBP (without potential IGF activation of IGF receptor signaling) • Determine the effects of the IGFBP on the cells mitogenic response to IGF-I • Establish if the Iguana IGFBP cell-surface associates by utilizing a RGD sequence

  10. IGF-I analogs [Ala31][Leu60]IGF-I Des(1-3)IGF-I IGF-I Cell surface IGFBP IGF-I receptor

  11. Materials and Methods IgH-2 cells Time and dose dependent removal of IGFBP IGF-I [Ala31][Leu60]IGF-I Des(1-3)IGF-I Affinity cross-linking of cell monolayer Western Ligand Blot of treatment media

  12. Materials and Methods Effects of the IGFBP on the cells mitogenic response to IGF-I Thymidine Incorporation assay • Two subsets of cells “ IGFBP present” and “IGFBP removed” • Treatment with IGF-I or Des(1-3)IGF-I • Addition of [3H]thymidine • Counted on LSC

  13. Materials and Methods IgH-2 cells Competitive inhibition of IGFBP cell membrane binding GRGDSP synthetic peptide GRGESP synthetic peptide Western Ligand Blot of treatment media Affinity cross-linking of cell monolayer

  14. Results • Time and dose dependent removal of the IGFBP from the cell surface

  15. 15 minute incubation with [Ala31][Leu60]IGF-I 0 1nM 3nM 0.1M 1M IGF Des IGF receptor (135 kDa) IGFBP bound with 125I-IGF-I (37 kDa) Released IGFBP (30 kDa) Rat Serum

  16. Results • Effect of the IGFBP on the cells mitogenic response to IGF-I

  17. Thymidine incorporation 1

  18. Results • Competitive inhibition of IGFBP cell surface association by a RGD synthetic peptide

  19. GRGDSP (M) RGE 0 25 50 100 500 IGF IGF receptor (135 kDa) IGFBP bound with 125I-IGF-I (37 kDa) Released IGFBP (30 kDa) Rat Serum

  20. Conclusions • IgH-2 IGFBP removal from the cell surface by IGF-I is time and dose dependent, but independent of IGF-I activation of receptor signaling functions to inhibit IGF-I growth stimulation cell surfaces associates through a RGD amino acid sequence • Future research will be directed towards the cloning of this IGFBP

  21. Acknowledgments I would like to thank: - Dr. Kevin Kelley, Kathleen Sak, Kameron Schmidt, and all the other members of the CSULB Endocrine Lab - Howard Hughes Medical Institute

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