Larissa Balogh Endocrine Laboratory California State University at Long Beach
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Transcript of Larissa Balogh Endocrine Laboratory California State University at Long Beach
The Growth Inhibitory Role and Cell The Growth Inhibitory Role and Cell Membrane Association of a Reptilian Membrane Association of a Reptilian
Insulin-Like Growth FactorInsulin-Like Growth Factor Binding Protein Binding Protein
Larissa BaloghLarissa Balogh
Endocrine Laboratory Endocrine Laboratory
California State University at Long BeachCalifornia State University at Long Beach
Research Advisor: Dr. Kevin M. KelleyResearch Advisor: Dr. Kevin M. Kelley
Insulin-like growth factorsInsulin-like growth factors
• Insulin-like growth factors (IGFs) are growth-Insulin-like growth factors (IGFs) are growth-stimulatory peptides stimulatory peptides
• IGF-I receptorIGF-I receptor activates cell growth activates cell growth
• Insulin receptor transduces metabolic functionsInsulin receptor transduces metabolic functions
INS/IGF Precursor(before vertebrate emergence)
INS IGF
X
IGFBP introduced
(early vertebrates?)
INS-Receptor IGF-Receptor(metabolic) (growth)
Cell Membrane
IGFBP?
Slide by Dr. Kevin Kelley
IGF-binding proteinsIGF-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
• Little is known about non-mammalian IGFBPsLittle is known about non-mammalian IGFBPs
• Six principal mammalian IGFBPsSix principal mammalian IGFBPs
• IgH-2 cell line
derived from heart epithelial cells
of the reptile, Iguana iguana
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
IgH-2 Iguana heart cellIgH-2 Iguana heart cell
HypothesisHypothesis
• The membrane-bound The membrane-bound IguanaIguana IGFBP IGFBP serves an IGF-inhibitory role in the heart serves an IGF-inhibitory role in the heart cells cells
IGF arriving at the cellular site is removed IGF arriving at the cellular site is removed from proximity to the growth receptorfrom proximity to the growth receptor
cell surface IGFBP
IGF-I receptor
GoalsGoals
• Determine the effects of IGF-I on cell-Determine the effects of IGF-I on cell-surface associated IGFBP (without potential surface associated IGFBP (without potential IGF activation of IGF receptor signaling)IGF activation of IGF receptor signaling)
• Determine the effects of the IGFBP on the Determine the effects of the IGFBP on the cells mitogenic response to IGF-Icells mitogenic response to IGF-I
• Establish if theEstablish if the Iguana Iguana IGFBP cell-surface IGFBP cell-surface associates by utilizing a RGD sequence associates by utilizing a RGD sequence
IGF-I analogsIGF-I analogs
IGF-I[Ala31][Leu60]IGF-I Des(1-3)IGF-I
Cell surface IGFBP
IGF-I receptor
Materials and MethodsMaterials and Methods
Time and dose dependent removal of IGFBPTime and dose dependent removal of IGFBP IgH-2 cells
IGF-I [Ala31][Leu60]IGF-I Des(1-3)IGF-I
Affinity cross-linking of cell monolayer
Western Ligand Blot of treatment media
Materials and MethodsMaterials and Methods
Thymidine Incorporation assayThymidine Incorporation assay
• Two subsets of cellsTwo subsets of cells“ IGFBP present” and “IGFBP removed”“ IGFBP present” and “IGFBP removed”
• Treatment with IGF-I or Des(1-3)IGF-I Treatment with IGF-I or Des(1-3)IGF-I
• Addition of [Addition of [33H]thymidine H]thymidine
• Counted on LSCCounted on LSC
Effects of the IGFBP on the cellsEffects of the IGFBP on the cellsmitogenic response to IGF-Imitogenic response to IGF-I
Competitive inhibition of IGFBP cell membrane binding
Materials and MethodsMaterials and Methods
IgH-2 cells
GRGDSP synthetic peptideGRGESP synthetic peptide
Western Ligand Blot of treatment media
Affinity cross-linking of cell monolayer
ResultsResults
• Time and dose dependent removal of the Time and dose dependent removal of the IGFBP from the cell surfaceIGFBP from the cell surface
Rat Rat SerumSerum
(135 kDa)(135 kDa)
(37 kDa)(37 kDa)
(30 kDa)(30 kDa)
Released Released IGFBPIGFBP
IGF receptorIGF receptor
IGFBP IGFBP bound with bound with 125125I-IGF-II-IGF-I
0 1nM 3nM 0.10 1nM 3nM 0.1M 1M 1M IGF DesM IGF Des
15 minute incubation 15 minute incubation with with [Ala[Ala3131][Leu][Leu6060]IGF-I]IGF-I
• Effect of the IGFBP on the cells mitogenic Effect of the IGFBP on the cells mitogenic response to IGF-Iresponse to IGF-I
ResultsResults
Thymidine incorporationThymidine incorporation
0.8
1
1.2
1.4
1.6
1.8
2
2.2
0 50 100
IGF-1 (ng / ml)
fold
sti
mu
lati
on
IGF-I IGF-I ([Ala][ Leu]IGF-I pretreated) Des(1-3)IGF-I
aa
a
b
c
a
1
ResultsResults
• Competitive inhibition of IGFBP cell Competitive inhibition of IGFBP cell surface association by a RGD synthetic surface association by a RGD synthetic peptidepeptide
Rat Rat SerumSerum
(135 kDa)(135 kDa)
(37 kDa)(37 kDa)
(30 kDa)(30 kDa)
Released Released IGFBPIGFBP
IGF receptorIGF receptor
IGFBP IGFBP bound with bound with 125125I-IGF-II-IGF-I
RGE 0 25 50 100 500 IGF RGE 0 25 50 100 500 IGF
GGRGDRGDSP (SP (M)M)
ConclusionsConclusions
• IgH-2 IGFBP IgH-2 IGFBP removal from the cell removal from the cell surface by IGF-I is time surface by IGF-I is time and dose dependent, but and dose dependent, but independent of IGF-I independent of IGF-I activation of receptor activation of receptor signaling signaling functions to inhibit IGF-I functions to inhibit IGF-I growth stimulationgrowth stimulation cell surfaces associates cell surfaces associates through a RGD amino through a RGD amino acid sequence acid sequence
• Future research will be directed towards the Future research will be directed towards the cloning of this IGFBP cloning of this IGFBP
AcknowledgmentsAcknowledgments
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