Download Insulin-like growth factors - California State University, 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
• Insulin-like growth factors (IGFs) are growthstimulatory peptides
• IGF-I receptor activates cell growth
• Insulin receptor transduces metabolic functions
INS/IGF Precursor
(before vertebrate emergence)
INS
IGFBP introduced
IGF
(early vertebrates?)
?
IGFBP
Cell Membrane
INS-Receptor
IGF-Receptor
(metabolic)
(growth)
Slide by Dr. Kevin Kelley
IGF-binding proteins
• IGF-binding proteins (IGFBPs) are the
integrators of the endocrine growthregulatory apparatus
block IGF interaction with insulin receptors
 regulate IGF bioactivity
IGF-independent effects
• 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
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
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
cell
surface
IGFBP
IGF-I
receptor
Goals
• Determine the effects of IGF-I on cellsurface 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
IGF-I analogs
[Ala31][Leu60]IGF-I
Des(1-3)IGF-I
IGF-I
Cell surface IGFBP
IGF-I receptor
Materials and Methods
Time and dose dependent
removal of IGFBP
IgH-2 cells
Western
Ligand
Blot of
treatment
media
IGF-I
[Ala31][Leu60]IGF-I
Des(1-3)IGF-I
Affinity
cross-linking
of cell
monolayer
Materials and Methods
Effects of the
IGFBP on the
cells
Thymidine
Incorporation
assay
mitogenic response to IGF-I
• 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
Materials and Methods
Competitive inhibition
IgH-2 cellsof IGFBP cell
membrane binding
Western
Ligand
Blot of
treatment
media
GRGDSP synthetic peptide
GRGESP synthetic peptide
Affinity
cross-linking
of cell
monolayer
Results
• Time and dose dependent removal of the
IGFBP from the cell surface
15 minute incubation
with [Ala31][Leu60]IGF-I
IGF receptor
(135 kDa)
0
1nM
3nM 0.1M 1M IGF
Des
IGFBP
bound with
125I-IGF-I
(37 kDa)
Released
IGFBP
(30 kDa)
Rat
Serum
Results
• Effect of the IGFBP on the cells mitogenic
response to IGF-I
Thymidine incorporation
IGF-I
IGF-I ([Ala][ Leu]IGF-I pretreated)
Des(1-3)IGF-I
2.2
c
fold stimulation
2
1.8
1.6
b
1.4
a
a
1.2
a
a
1
0.8
0
1
50
IGF-1 (ng / ml)
100
Results
• Competitive inhibition of IGFBP cell
surface association by a RGD synthetic
peptide
GRGDSP (M)
IGF receptor
(135 kDa)
RGE
0
25
50
100
500
IGF
IGFBP
bound with
125I-IGF-I
(37 kDa)
Released
IGFBP
(30 kDa)
Rat
Serum
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
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