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Effects of pro-TGFα on EGF receptor signaling

Hindupur Vasantamadhava, Sruthi (2014) MOBM15 20141
Degree Projects in Molecular Biology
Abstract
TGFα is a member of the epidermal growth factor (EGF) family and is one of the ligands for the EGF- receptor. The precursor of TGFα, pro-TGFα, is a transmembrane protein. The aim of this project has been to establish a model for studies of pro-TGFα effects on EGF receptor signalling, in particular through Gab1, a docking protein involved in PI3K/Akt signalling pathway. The experiments in this thesis were a part of pilot studies and were performed in HeLa cells. HeLa cells were transfected with wild type pro-TGFα and a mutated pro-TGFα which lacks the cytoplasmic domain except for the terminal 4 residues. The transfected cells were studied using western blotting, fluorescence microscopy and confocal microscopy. Results showed transfection... (More)
TGFα is a member of the epidermal growth factor (EGF) family and is one of the ligands for the EGF- receptor. The precursor of TGFα, pro-TGFα, is a transmembrane protein. The aim of this project has been to establish a model for studies of pro-TGFα effects on EGF receptor signalling, in particular through Gab1, a docking protein involved in PI3K/Akt signalling pathway. The experiments in this thesis were a part of pilot studies and were performed in HeLa cells. HeLa cells were transfected with wild type pro-TGFα and a mutated pro-TGFα which lacks the cytoplasmic domain except for the terminal 4 residues. The transfected cells were studied using western blotting, fluorescence microscopy and confocal microscopy. Results showed transfection efficiencies close to 30 and 40%, respectively, for the two probes. The wild type pro-TGFα was found at the plasma membrane and reduced surface expression of mature TGFα was noticed in cells transfected with the mutated form. Ectopic expression of either pro-TGFα forms induced increased Gab1 and caveolin-1 activation, whereas the wild type pro- TGFα surprisingly reduced EGFR phosphorylation on several autophosphorylation sites. In further experiments involving confocal microscopy, significant pro-TGFα – Gab1 and pro-TGFα-EGFR colocalization was observed towards the cell periphery. Results suggested that pro-TGFα may play a crucial role in EGFR signalling, and may be necessary for Gab1 activation. However, the obtained results are not conclusive and there were problems involving the methodology. More experiments need to be performed to clearly understand the role of pro-TGFα in EGFR signalling. (Less)
Popular Abstract
Cancer is a complex disease involving over expression of growth factor receptors and growth factors, oncogene activation, tumour-suppressor gene inactivation and dysfunctional intracellular signalling pathways. Hence it is of high significance to study the roles of signalling pathways and various signalling proteins to understand the mechanisms that can lead to the disease and to diagnose and treat the disease at an early stage.

The large family of Receptor Tyrosine Kinases (RTK) which are present on the cell surface are major role players in cell signalling. EGFR is an extensively studied RTK which plays an important role in signalling pathways involved in growth, signalling, migration and survival of both normal and cancer cells.... (More)
Cancer is a complex disease involving over expression of growth factor receptors and growth factors, oncogene activation, tumour-suppressor gene inactivation and dysfunctional intracellular signalling pathways. Hence it is of high significance to study the roles of signalling pathways and various signalling proteins to understand the mechanisms that can lead to the disease and to diagnose and treat the disease at an early stage.

The large family of Receptor Tyrosine Kinases (RTK) which are present on the cell surface are major role players in cell signalling. EGFR is an extensively studied RTK which plays an important role in signalling pathways involved in growth, signalling, migration and survival of both normal and cancer cells. EGFR family receptors are activated by multiple ligands of which both EGF and TGFα bind to the receptor with high affinity. TGFα is synthesised as 160 aminoacid long precursor which then transforms into pro-TGFα which is transported to cell membrane and mature TGFα is released after metalloprotease cleavages.

The aim of this project has been to establish a model for studies of pro-TGFα effects on EGF receptor signalling, in particular through Gab1, a docking protein involved in one of the pathways activated by EGFR. Two different forms of pro-TGFα, a wildtype and one with a deleted cytoplasmic tail, were transfected into HeLa cells to analyse the effects on signalling using fluorescence microscopy, confocal microscopy and western blotting techniques. It was observed that the mutated pro-TGFα was expressed low on the cell surface. There was reduced activation of EGFR on various sites in cells with wildtype pro-TGFα and also sustained Gab1 activation. The confocal microscopy images suggested that pro- TGFα and Gab1 may be located in close compartments in the cell. This suggests that pro-TGFα may play a role in Gab1 activation through an alternate mechanism. However the results were not sufficient to conclude the role of pro-TGFα in EGFR signalling but create a sound basis for a more conclusive investigation further and hopefully revealing novel signalling mechanism.

Supervisor: Henrik S. Huitfeldt
Master´s Degree Project - Cell and Molecular Biology, 30 credits. 2014
Dept. Pathology, Oslo University Hospital, Oslo
Lund University, Department of Biology (Less)
Please use this url to cite or link to this publication:
author
Hindupur Vasantamadhava, Sruthi
supervisor
organization
course
MOBM15 20141
year
type
H2 - Master's Degree (Two Years)
subject
language
English
id
4863880
date added to LUP
2014-12-15 15:11:46
date last changed
2014-12-15 15:11:46
@misc{4863880,
  abstract     = {TGFα is a member of the epidermal growth factor (EGF) family and is one of the ligands for the EGF- receptor. The precursor of TGFα, pro-TGFα, is a transmembrane protein. The aim of this project has been to establish a model for studies of pro-TGFα effects on EGF receptor signalling, in particular through Gab1, a docking protein involved in PI3K/Akt signalling pathway. The experiments in this thesis were a part of pilot studies and were performed in HeLa cells. HeLa cells were transfected with wild type pro-TGFα and a mutated pro-TGFα which lacks the cytoplasmic domain except for the terminal 4 residues. The transfected cells were studied using western blotting, fluorescence microscopy and confocal microscopy. Results showed transfection efficiencies close to 30 and 40%, respectively, for the two probes. The wild type pro-TGFα was found at the plasma membrane and reduced surface expression of mature TGFα was noticed in cells transfected with the mutated form. Ectopic expression of either pro-TGFα forms induced increased Gab1 and caveolin-1 activation, whereas the wild type pro- TGFα surprisingly reduced EGFR phosphorylation on several autophosphorylation sites. In further experiments involving confocal microscopy, significant pro-TGFα – Gab1 and pro-TGFα-EGFR colocalization was observed towards the cell periphery. Results suggested that pro-TGFα may play a crucial role in EGFR signalling, and may be necessary for Gab1 activation. However, the obtained results are not conclusive and there were problems involving the methodology. More experiments need to be performed to clearly understand the role of pro-TGFα in EGFR signalling.},
  author       = {Hindupur Vasantamadhava, Sruthi},
  language     = {eng},
  note         = {Student Paper},
  title        = {Effects of pro-TGFα on EGF receptor signaling},
  year         = {2014},
}