How would you design an experiment to test whether M-CSF growth factor receptor signaling is dependent on dimerization of the receptor? Solution Answer: M-CSF growth factor receptor is a member of a family of homodimeric cytokines that share some sequence and structural similarity. Binding of its cognate bivalent ligands by this class of receptors stabilizes their non-covalent dimerization, permitting receptor activation and trans- tyrosine phosphorylation of one ICD (Intra-cellular domain) by the other. Once the growth factor has bound to and activated the receptor, newly phosphorylated tyrosine residues on the intracellular carboxy terminus of the receptor serve as binding sites for cytosolic or membrane-associated proteins that contain phosphotyrosine-binding domains. The best characterized of these domains are the rc-omology 2 (SH2) domains that share characteristic features first described in the phosphotyrosine-binding region of the cytosolic tyrosine kinase. Mona/Gads is a Grb2-related, Src homology 3 (SH3) and SH2 domain-containing adapter protein whose expression is restricted to cells of hematopoietic lineage. It is known to interact with the phosphorylated Y697 site of the M-CSFR. A simple experment to test that M-CSF growth factor receptor signaling is dependent on dimerization of the receptor is as follows: 1. Preparation of a WT (which can dimerize) and mutant version of M-CSF receptor such that it cannot dimerize. This can be done through over expression of the protein in insect cells (Sf9 cells) with FLAG-tag and judging for the oligomeric state through size exclusion chromatography (FPLC). (The insect cells sholud be co-infected with the kinase baculovirus as well, if the insect cells doesn\'t contain the same. This would facilitate phosphorylation of ICD) 2. Both the receptors can be purified and a Co-Immunoprecipitation (Co-IP) experiment can be performed with purified Mona/Gads or with cell extracts expressing the same. 3. After elution, a western blot can be performed and probed for the FLAG-tagged receptors. The dimerized WT receptor should bind to Mona/Gads and appear as bands in the western blot after developing, whereas the monomeric form should not show up in the blot. This would prove that Mana/Gads binds to dimeric receptor as compared to the monomeric form and facilitate the downstream signaling cascades. Similar experiments can be performed in-vivo, such as in HEK293 or HeLa cells by overexpressing tagged receptor and Mona/Gads and probing for the binding through western blot..

1. How would you design an experiment to test whether M-CSF growth factor receptor signaling is
dependent on dimerization of the receptor?
Solution
Answer:
M-CSF growth factor receptor is a member of a family of homodimeric cytokines that share
some sequence and structural similarity. Binding of its cognate bivalent ligands by this class of
receptors stabilizes their non-covalent dimerization, permitting receptor activation and trans-
tyrosine phosphorylation of one ICD (Intra-cellular domain) by the other.
Once the growth factor has bound to and activated the receptor, newly phosphorylated tyrosine
residues on the intracellular carboxy terminus of the receptor serve as binding sites for cytosolic
or membrane-associated proteins that contain phosphotyrosine-binding domains. The best
characterized of these domains are the rc-omology 2 (SH2) domains that share characteristic
features first described in the phosphotyrosine-binding region of the cytosolic tyrosine kinase.
Mona/Gads is a Grb2-related, Src homology 3 (SH3) and SH2 domain-containing adapter
protein whose expression is restricted to cells of hematopoietic lineage. It is known to interact
with the phosphorylated Y697 site of the M-CSFR.
A simple experment to test that M-CSF growth factor receptor signaling is dependent on
dimerization of the receptor is as follows:
1. Preparation of a WT (which can dimerize) and mutant version of M-CSF receptor such that it
cannot dimerize. This can be done through over expression of the protein in insect cells (Sf9
cells) with FLAG-tag and judging for the oligomeric state through size exclusion
chromatography (FPLC). (The insect cells sholud be co-infected with the kinase baculovirus as
well, if the insect cells doesn't contain the same. This would facilitate phosphorylation of ICD)
2. Both the receptors can be purified and a Co-Immunoprecipitation (Co-IP) experiment can be
performed with purified Mona/Gads or with cell extracts expressing the same.
3. After elution, a western blot can be performed and probed for the FLAG-tagged receptors. The
dimerized WT receptor should bind to Mona/Gads and appear as bands in the western blot after
developing, whereas the monomeric form should not show up in the blot.
This would prove that Mana/Gads binds to dimeric receptor as compared to the monomeric form
and facilitate the downstream signaling cascades. Similar experiments can be performed in-vivo,
such as in HEK293 or HeLa cells by overexpressing tagged receptor and Mona/Gads and
probing for the binding through western blot.