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 peptidesInsulin-like growth factors (IGFs) are growth- stimulatory peptides IGF-I receptor activates cell growthIGF-I receptor activates cell growth Insulin receptor transduces metabolic functionsInsulin receptor transduces metabolic functions

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

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

5. Hypothalamus GRH Anterior Pituitary GH Liver IGF IGFBP IGF IGFBP

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

7. single IGFBP, binds 125 I-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 IgH-2 Iguana heart cell

8. Hypothesis The membrane-bound Iguana IGFBP serves an IGF-inhibitory role in the heart cellsThe 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

9. cell surface IGFBP IGF-I receptor

10. Goals Determine the effects of IGF-I on cell- surface associated IGFBP (without potential IGF activation of IGF receptor signaling)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-IDetermine 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 sequenceEstablish if the Iguana IGFBP cell-surface associates by utilizing a RGD sequence

11. IGF-I analogs IGF-I [Ala 31 ][Leu 60 ]IGF-IDes(1-3)IGF-I Cell surface IGFBP IGF-I receptor

12. Materials and Methods Time and dose dependent removal of IGFBP Time and dose dependent removal of IGFBP IgH-2 cells IGF-I [Ala 31 ][Leu 60 ]IGF-I Des(1-3)IGF-I Affinity cross-linking of cell monolayer Western Ligand Blot of treatment media

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

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

15. Results Time and dose dependent removal of the IGFBP from the cell surfaceTime and dose dependent removal of the IGFBP from the cell surface

16. Rat Serum (135 kDa) (37 kDa) (30 kDa) (30 kDa) Released IGFBP IGF receptor IGFBP bound with 125 I-IGF-I 0 1nM 3nM 0.1  M 1  M IGF Des 0 1nM 3nM 0.1  M 1  M IGF Des 15 minute incubation with [Ala 31 ][Leu 60 ]IGF-I

17. Effect of the IGFBP on the cells mitogenic response to IGF-IEffect of the IGFBP on the cells mitogenic response to IGF-I Results

18. Thymidine incorporation 1

19. Results Competitive inhibition of IGFBP cell surface association by a RGD synthetic peptideCompetitive inhibition of IGFBP cell surface association by a RGD synthetic peptide

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

21. 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 sequenceIgH-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 IGFBPFuture research will be directed towards the cloning of this IGFBP

22. 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