Lecture 2 - Regulation of protein expression (cytokine signalling)

Question 1

Range of responses a target cell can have to a ligand

Answer 1

Grow, divide, die, differentiate, become active (contract, move, action potential, release a signal of its own, make more of something, breakdown more of something)

All of these responses involve changing the AMOUNT or ACTIVITY of a protein in response to the signal

Question 2

Role of protein expression and activity in responses

Answer 2

To change the way a cell is behaving is to change the make of a particular protein or a group of proteins

Increasing the gene expression increases the transcription and translation of that gene which means you get more of the protein that the gene encodes for

Proteins do not do much as just a naive protein, in order to become active they need to become activated (they needed to be turned from an inactive shape into an active shape) (proteins can also be deactivated to stop them from producing a particular response

Cell signalling pathways act on… 
Gene —> protein pathway (change expression) 
Protein  activation (change activity) 
Cell signalling pathways act here to change activity and/or expression (amount) of a protein

Question 3

Different types of cell surface receptors

Answer 3

Ion channel, G protein, cytokine

These 3 are all receptors that sit on the cell surface and their job is to detect molecules that are water soluble because being water soluble means that they cannot cross the phospholipid membrane so they must interact with receptors on the cell surface and these receptors have different structures

-Signals transduced into the cell through ion channels and G proteins have cytoplasmic effects. Cytokine receptors have effects on the nucleus

Question 4

Ion channel

Answer 4

Ion channels are pore-forming membrane proteins that allow ions to pass through the channel pore.

Question 5

G protein

Answer 5

Ligand binds, signal is transduced to inside the cell using a G protein

Question 6

Steroid

Answer 6

Not cell surface but intracellular = steroid
Receptor is inside the cell and therefore it can receive lipid soluble ligand signals such as steroids as they can move across the phospholipid membrane

Question 7

Cytokines

Answer 7

A broad range of cell signalling molecules

Cytokines are basically proteins, peptides and glycoproteins that signal from one cell to another (intercellular signalling)

Involved in autocrine, paracrine and endocrine signalling

Originally associated with the immune system, signals to many cells in the immune system but also to other cells

Includes interferons (IFN) and interleukins (IL) plus additional factors

Cytokines act via cell surface cytokine receptors - there are multiple subtypes with similar mechanisms

Question 8

Importance of cytokine signalling

Answer 8

Involved in autocrine, paracrine and endocrine signalling

Question 9

Growth hormone

Answer 9

Peptide hormone (short protein), also called somatotropin, is produced by somatotrophin cells in the anterior pituitary

It stimulates the growth of essentially all tissues of the body, including bone

GH has anabolic effects on the body e.g. it causes tissue to grow like the muscle and bone

Many GH actions are secondary to its ability to stimulate insulin-like growth factor (IGF-1) release from the liver

Question 10

Feedback system of GH

Answer 10

Release increased by GH-releasing hormone from the hypothalamus and ghrelin from the stomach
Ghrelin is produced in response to eating a lot of food which is a message to your anterior pituitary to start making growth hormone because you have just had an intake of food
GH releasing hormone is released into a blood supply that connects the hypothalamus and anterior pituitary

Release is inhibited by GH-inhibiting hormone also from the hypothalamus
GHRH stimulates the release of GH from the anterior pituitary
Somatostatin inhibits the release of GH from the anterior pituitary.
GH feeds back to the hypothalamus preventing the release of more GH (negative feedback)
Somatomedin C/IGF1 produced by the liver feeds back to inhibit GHRH and promote somatostatin release from the hypothalamus

Question 11

Mechanisms of GH receptor signalling

Answer 11

GH binds to the GH receptor on the surface of target cells, including the hepatocytes in the livers . Causes two GH receptors to come together

GH binds to two receptor monomers which join together i.e. it causes them to dimerise
This dimerised receptor complex becomes active because the associated JAK2 protein come into contact with each other - the JAK2 protein on the left puts a phosphate on the right one and vice versa
JAK is a protein tyrosine kinase and when activated adds phosphates to intracellular signalling molecules including STAT5 and ERK1/2

Once JAK2 becomes active then they can affect other molecules in the cell - one of the molecules they affect is called STAT5 which becomes phosphorylated and once this occurs it can go into the nucleus and turn on the transcription of particular genes.ERK1/2 which can also be phosphorylated can also act as a transcription factor like STAT5

Phosphorylated STAT5 and ERK1/2 enter the nucleus and act as transcription factors turning on (or off) specific genes. In the case of hepatocytes this would include increasing the production of IGF-1

Question 12

Summary of the four main steps in immunohistochemistry

Answer 12

Tissue section expressing antigen (e.g. STAT5)

Addition of primary antibody which binds to antigen

Addition of secondary antibody which binds to primary antibody

Enzyme on secondary antibody allows visible staining to occur

Question 13

STAT5 part of the GH signalling pathway

Answer 13

STAT5 exists as two closely related forms STAT5a and STAT5b

When phosphorylated by JAK2, phospho-STAT5 may form homo or heterodimers
Homo = stat5a and a OR hetero = stat5a and b

When they become phosphorylated and dimerised, STAT dimer moves into the nucleus via nuclear pore.

Once in the nucleus the STAT5 dimers interact with another transcription factor which is activated by glucocorticoids.
Once in the nucleus, can get signals from one of the steroids such as glucocorticoids which connects on its own receptor and the GR (glucocorticoid receptor) and the STAT5 receptor get together and the combination of the activity of these two things is how genes get turned on and off

Intracellular negative feedback pathways now turn off the phosphoSTAT5 response

Other branches of the growth hormone signalling pathway regulate cytoplasmic events

Question 14

STATs stands for …

Answer 14

(Signal Transducers and Activators of Transcription)