MP4: Cell Signalling Flashcards
State the 6 general steps in a signaling pathway
- triggering of response
- transfer of information across a membrane
- amplification of signal
- location of signal
- divergence of one signal to a range of response proteins
- termination of response
State the different modes of signaling between cells
- diffusible signal, long range
- diffusible signal, short range (paracrine and autocrine)
- signaling by cell contact
- signaling by cytoplasmic connection
Define: paracrine
acts on something nearby
Define: autocrine
signals act on the same cell producing them (harder to terminate the signal compared to paracrine)
Define: chemotaxis
the phenomenon by which cell movement is directed by a molecular concentration gradient (e.g., B and T cells)
Define: secondary messengers
molecules released into the cytosol in response to the primary signal outside of the cell
Explain the process of adding and removing a phosphate PTM
These are reversible PTMs in which kinases transfer the terminal phosphate group from ATP onto an amino group of a protein, releasing an ADP. This will be specific and defined by the amino acids surrounding the residue to be phosphorylated. Phosphatases will then cleave the phosphate group off when the signal needs to be changed.
Describe the advantages of phosphorylation
- rapid
- direct use of cellular energy source
- addition specifically catalysed by an enzyme - kinase
- reversible but requires an enzyme to remove the phosphate - phosphatase
- both enzymes show specificity
Give the 3 key sites of phosphorylation
- amino acid side chains with OH (e.g. serine and threonine - sometimes tyrosine)
- lower eukaryotes also have histidine phosphorylation
- OH groups on lipids e.g., inositol ring on the headgroups of phospholipids
What can SH2 domains interact with? Give an example pathway.
Phospho-tyrosine residues can interact with these domains on proteins. e.g., PIP2 —> IP3 + DAG pathway via autophosphorylation of the tyrosine kinase receptor
Describe PH domains
These can interact with inositol lipids e.g., PIP2 —> PIP3 which releases PH-PKB(hydroxyl)
Describe the addition and removal of G proteins
The protein will contain a binding site for the guanine nucleotide (either GTP or GDP). If inactive, GDP is bound. Guanine nucleotide exchange factor (GEF) allows the GDP to diffuse out and replaces it with GTP, causing a conformational change in the protein and its activation. To terminate the signal, the proteins have intrinsic GTPase activity to remove the GTP, but GTPase activating proteins (GAPs) can assist.
What are the two classes of G proteins? Describe their structures.
- small monomeric GTPase proteins
- single subunit
- conserved GTPase domain - heterotrimeric G proteins
- 3 subunits: alpha, beta, and gamma
- alpha subunit is GTPase
- activated by GPCRs
2. heterotrimeric G proteins
Describe one component systems and give examples
Input and output are on the same molecule, driving conformational change that changes the ability to bind to either DNA or RNA polymerase.
E.g., the lac repressor (allolactose)/Trp repressor (tryptophan)/CAP (cAMP)/FNR (oxygen)
Describe two component systems and give examples
Involves two molecules such that there is an input molecule and an output molecule.
E.g., the Ntr system and the chemotaxis system in control of the flagellum motor: sensor is histidine protein kinase and output is the response regulator protein
Give the location and describe the structure of most histidine protein kinases.
- most are membrane proteins, but all are dimers that undergo transautophosphorylation
- the bond formed between the phosphate and the histidine is easily hydrolyzed and thus unstable
- contains three key domains:
1. N-terminal sensor domain
2. DHp domain that contains the phosphorylated histidine and is responsible for dimerization
3. C-terminal kinase domain
Describe response regulator proteins.
A response regulator is a protein that mediates a cell’s response to changes in its environment as part of a two-component regulatory system. Response regulators are coupled to specific histidine kinases which serve as sensors of environmental changes. Response regulators and histidine kinases are two of the most common gene families in bacteria, where two-component signaling systems are very common.
- aspartate becomes phosphorylated, by removing the phosphoryl group from the unstable histidine kinase
- phosphoanhydride bond (not phosphoester) which is also unstable
- majority have two domains:
1. Receiver (REC) that contains the aspartate
2. Either DNA binding domain (69%) or enzymatic domain (8%)
Give an overview of G protein coupled receptors. Include structure and how they transmit a signal through the membrane.
- 7 transmembrane domains
- Compact protein with relatively small movements in helices 5 and 6. These can rotate around one another, leading to conformational changes that cross the membrane to transmit the signal
- Loop between 5 and 6 interacts with the G protein, thus the GPCR acts as a GEF
What is the structure of tyrosine kinase receptors? How do they transmit a signal? What proteins bind them?
Give an example pathway.
- single TM domain and intrinsic TK activity at the C-terminal
- form dimers upon ligand binding, allowing for transphosphorylation of tyrosines in the C-terminus
- SH2 or PTB domain proteins can bind phosphorylated tyrosines (also dependent on the surrounding amino acids)
Example pathway: ligand binds TK receptor –> autophosphorylation –> Grb2 (adaptor protein) binds via SH2 domain —> SH3 domain of Grb2 binds to SOS protein. SOS is a GEF which activates Ras by exchanging GDP for GTP. Once GTP-bound, Ras can trigger transmission of the signal.
What are scaffold proteins?
Bind several proteins within a cascade, localising the signal and providing specificity but limits amplification
What is the function of PI3 kinase? What domains does it contain? How does it interact with PKB? Give an example pathway that it’s used in.
- lipid kinase that phosphorylates the 3’ position of inositol rings (e.g., PIP2 into PIP3)
- contains SH2 domains and PP domains (which interact with SH3)
- PI3K activates PKB via phosphorylation of serine and threonine residues (activation also requires phosphorylation by PDK1 and mTOR2)
Example pathway: cell growth
- IGF activates TK receptor
- IRS1 binds SH2; PI3K binds SH3 of IRS1
- PI3K cleaves PIP2 into PIP3
- PIP3 binds Akt and PDK1
- Akt allows for mTORC1 to phosphorylate further proteins and increase protein synthesis for more growth
What is the function of mTORC? What inhibits/regulates mTORC1 and how?
A protein kinase complex that’s implicated in cell growth and metabolism downstream of PKB and also of nutrient status e.g., amino acids to integrate complex growth responses.
mTORC1 is inhibited by a complex of rapamycin and FKBP12.
It’s regulated by amino acids on lysosomes.
What property allows most signals to travel in the bloodstream? How is this overcome for hydrophobic signals?
Most signals are hydrophilic to be able to travel in the bloodstream.
Some are hydrophobic, and so they tend to be made close to their target site to prevent movement over long distances.
Give an example where the same signaling molecule can be used to trigger 3 different pathways.
Acetylcholine can trigger:
1. decreased firing rate in heart pacemaker cells
2. secretion in salivary gland cells
3. contraction in skeletal muscle cells
How can kinase activity be monitored?
- Immunoprecipitate the kinase of interest
- Mix with protein of interest for different time periods.
- Western blot for each time period
- Use a phosphospecific antibody for the substrate and compare to another WB that only shows the amount of protein present from each time period.
How can chimeric receptor experiments show specificity of GPCRs and heterotrimeric G proteins?
Chimeric receptors are made by swapping intracellular portions at the level of the gene. When these chimeric receptors are expressed in cells, they activate the ‘wrong’ pathway in response to a ligand.
This showed that specificity is defined by the 5-6 intracellular loop.
What are the two functions of B-arrestin?
In response to the activation of a GPCR, beta-arrestin is recruited to the receptor and binds its phosphorylated intracellular domain. This binding event causes the receptor to be internalized and desensitized, leading to the termination of the initial signaling response.
It can also recruit other signaling proteins to the receptor, such as MAPKs and Src kinases.
