Cell Signalling, (Chapt. 13 in Cooper).

Cells communicate with one another via a wide variety of different "signal transduction" mechanisms, in many ways analogous to human communication via speech, sight, writing, phone, email, etc. While signal transduction pathways have been studied for many years, mostly by physiologists and pharmacologists, it is only in the last 10-20 years that we have begun to understand many of these at the molecular level, due to the ability to identify and isolate the biochemical components involved and the genes which encode relevant protein participants. Most signal transduction processes involve a series of steps during which energy/information may change forms (transduce) several times (e.g., compare to talking on the phone).

One way to classify types of signal transduction processes (Fig. 13.1):

1. Endocrine: transmitter carried to distant sites via circulatory system (e.g., steroid hormones, email)

2. Paracrine: transmitter can reach only nearby cells or sometimes only those in direct contact (e.g., acetylcholine at nerve/muscle synapse, giving a lecture)

3. Autocrine: transmitter affects same cell that makes it (e.g., immune cells that synthesize their own growth factors, memo to myself)

General Principles and Definitions: With the exception of membrane depolarization, discussed earlier in the course, most cell signalling involves the generation by an effector cell of a molecular transmitter (hormone or factor), whose properties are dependent on diffusion rate, solubility, and binding to cell proteins/structures. The protein (or other structure) of the affected cell which binds the transmitter is called a receptor. Occasionally the receptor directly initiates the desired response in the affected cell, but more often the receptor transduces the signal through its effect on intracellular proteins, DNA, or a small molecule. The latter is often called a second messenger. Thus, generally a series (or cascade) of steps is involved, which provides opportunities for amplification of the signal (e.g., pyramid scheme) and integration of different signals at common points (e.g., voting booth). Since almost all biological processes are cyclical, virtually all signalling processes turn off as well as turn on. The turning off process may (rarely) involve spontaneous decay, but it is often regulated by another set of effector proteins/enzymes and the off process may initiate a signal as well as the on process.


Some prominent transmitters: How the message gets to the target cell.

Steroid Hormones (Fig. 13.2-13.4) include glucocorticoids, thyroid hormone, testosterone, retinoic acid, and vitamin D3, among others. They are lipid in character (made from cholesterol) and can freely diffuse across cell membranes, so steroid hormone receptors (proteins) exist in the cytoplasm and nucleus of affected cells. Steroid hormone receptors are transcriptional regulators that bind to DNA and turn adjacent genes on or off (or both). Binding to the hormone can alter the DNA binding properties of the receptor or its transcriptional activation properties (or both). Response to steroid hormones requires changes in gene expression, so their effects usually take hours to days.

Nitric Oxide (NO) is a paracrine transmitter (short half life). Effector cells synthesize NO which diffuses rapidly (dissolved gas) across membranes to nearby cells to alter activities of key enzymes in the affected cell (e.g., increased second messenger cGMP production by guanylyl cyclase). Among other things, NO is involved in blood vessel dilation of endothelial cells; reponse is in seconds or less.

Neurotransmitters (Fig. 13.6) are small hydrophilic molecules released from effector cells in response to membrane depolarization. Generally paracrine and/or endocrine. Diffuse rapidly but not across membranes. Bind cell surface receptors--often bind to and gate ion channel proteins discussed earlier in the course or to G-protein coupled receptors (see below).

Peptide Hormones, Neuropeptides, and Growth Factors. (Table 13.1) Can be endocrine, paracrine or autocrine. Canít diffuse across membranes, so they bind to specific cell surface receptors (see below).


Cell Surface Receptors: How signals cross the cell membrane.

Cell surface receptors function to transduce a signal from a molecule outside a cell across the cell membrane to the appropriate targets within the cell. They are generally integral membrane proteins (or protein complexes) with transmitter binding sites outside the cell, a -helical regions crossing the cell membrane and a cytoplasmic domain that generates a signal inside the cell upon transmitter binding outside the cell (due to conformational change, dimerization). There are several major families of such receptors. Members of each family generally differ in their transmitter binding sites and sometimes in aspects of the way their cytoplasmic domains transduce the signal to the inside of the cell.

1. Gated Ion Channel Receptors (Fig. 12.23) such as the acetylcholine receptor have been described earlier in the course. Receptor binding triggers opening (or closing) of the ion channel and changes the electrical properties of the cell membrane. Most heaviliy involved in nerve and muscle cell signalling.

2. G Protein-Coupled Receptors (Figs. 13.10-13.12) contain 7 membrane spanning a -helices. These receptors associate with trimeric G proteins bound to the inside of the cell membrane. The G proteins consist of three subunits, a , b , and g . The a subunit binds GDP or GTP. Receptor binding triggers a conformational change which causes the a subunit to separate from the b g subunits and to exchange its GDP for GTP. Both the Ga -GTP and free Gb g complexes can diffuse along the membrane and either stimulate or inhibit their targets. The targets may either be enzymes such as adenylyl cyclase which generates the second messenger cAMP (Fig. 13.11) or channels which induce membrane depolarization. When GTP on Ga is hydrolyzed to GDP and Pi, it reassociates with Gb g and the "switch" is turned off.

3. Receptor Tyrosine Kinases (Fig13.14, 13.15) generally contain a single membrane spanning helix, an extracellular binding domain and an intracellular protein kinase domain. Upon binding, the receptors dimerize, which allows the two chains to phosphorylate one another on specific tyrosine residues. This leads to the binding of proteins which have "SH2 domains", peptide regions that bind to phosphorylated tyrosines. These SH2 proteins may become phosphorylated and activated in different ways. Many are themselves protein kinases that go on to phosphorylate other signal proteins. Tyrosine kinase signalling is turned off by protein tyrosine phosphatases, which may themselves be signal-regulated, as will be seen below.

4. Cytokine receptors-Non-receptor tyrosine kinases (Fig. 13.17). (Cytokines are cell growth factors, especially those involved in white and red blood cell development.) Cytokine-type receptors are much like receptor tyrosine kinases, except they lack kinase activity themselves. They function by binding a non-receptor tyrosine kinase, thereby doing the same job with two separate proteins.

How do we know?

1. About receptors? Receptor proteins tend to be rare and those in membranes can be especially difficult to purify (like most integral membrane proteins). Many receptors were initially identified by on their ability to bind radioactive transmitters or drug analogues (estrogen, acetylcholine, etc.; usually some sort of filter binding assay is used in which the receptor sticks, but unbound hormone does not). Such an assay allows one to seek the cells or tissues that are the best source of receptor and then to purify minute quanitities of it. The next step is often to use partially purified receptor protein as an antigen to make monoclonal antibodies to the protein (see p. 117). Monoclonal antibodies are made by selecting immortalized mouse cells that grow in tissue culture and produce a particular antibody molecule that binds to a single site on a specific protein or other antigen. Since one can grow as many cells as one can afford, lots of antibody can be produced (and it can be labeled for detection in various ways). For example, the groups of Shrader and O'Malley and of Chambon made monoclonal antibodies against the chicken progesterone (steroid hormone) receptor. They then used an expression cDNA library made from RNA from chick oviduct, a tissue known to make the receptor. Double-stranded cDNA (Fig. 3.20) was made from the RNA and inserted into a recombinant DNA vector that expressed the inserted sequence as a protein (Fig. 3.26). [Usually only a part of the protein is made, but that is all that's needed for antibody binding.] Then a library is screened to identify those bacteria that make protein that binds labeled monoclonal antibody. Recombinant plasmids or phage from those bacteria should contain at least part of the receptor cDNA which can then be used to get the whole gene.

Intracellular signal pathways: how the message moves around the cell.

1. The second messenger cAMP. (Fig. 13.18-13.21) As noted above, G protein coupled signalling can activate adenylyl cyclase, causing intracellular cyclic AMP (cAMP) to increase. cAMP is a second messenger within the cell which can have numerous effects. [cGMP can also act as a second messenger, and both cAMP and cGMP levels can be controlled both by triggering degradation (phosphodiesterases) as well as synthesis (cyclases)]. Two major examples:

A. Protein kinase A is a serine kinase activated by cAMP-mediated dissociation of inhibitory subunits. Protein kinase A phosphorylates glycogen synthase to inactivate it and phosphorylase kinase to activate it. Phosphorylase kinase then phosphorylates glycogen phosphorylase to activate it to degrade glycogen to provide metabolic energy. Note that there is a dual switch, both turning on glycogen degradation and turning off glycogen synthesis. Note also that a "cascade" of two protein kinases is involved.

B. Protein kinase A, once activated, can also go to the nucleus and phosphorylate the transcription factor CREB (cAMP response element binding protein). Phosphorylated CREB turns on genes that have a CRE DNA sequence in their promoter regions.

2. Inositol triphosphate (IP3) and diacylglycerol (DAG) are two second messengers produced by the same reaction (Fig. 13.24-25). Both G protein receptors and tyrosine kinase receptors can activate forms of the enzyme phospholipase C which cleaves the membrane phospholipid phosphatidyl inositol 4,5 bisphosphate (PIP2) into IP3 and DAG (hereís an example of another dual action switch).

A. DAG activates protein kinase C (ser/thr kinase, Fig. 13.27) which activates gene-specific transcription factors either by directly phosphorylating them or by triggering kinase cascades.

B. IP3 diffuses into the cytoplasm and triggers release of Ca++ from the lumen of the endoplasmic reticulum (Fig. 13.28). Ca++ is also a second messenger (or in this case a third messenger).

3. MAP kinase pathways are protein kinase cascades triggered by the Ras protein or its analogues (Fig. 13.32-13.36). Different cells may utilize a variety of different forms of MAP kinase (mitogen activated protein kinase) pathways (Fig. 13.36). We will consider only the common elements of all pathways, with a few examples.

A. The initial mitogenic (cell growth promoting) signal is received by cell surface receptors and coupled to the activation of Ras or a Ras-type protein. (Fig. 13.34 gives one such example involving Grb2 and SOS; you donít need to know these two proteins.)

B. Ras proteins are monomeric guanine nucleotide binding proteins (similar to Ga proteins, but with no other subunits). They are active when bound to GTP and inactive when bound to GDP. They have GTPase activity, so they have an intrinsic off rate (like an iron that turns itself off when not used for awhile). Ras proteins provide a key control point as they can be activated or inactivated by several other proteins (Fig. 13.33).

C. In their active (GTP-bound) state, Ras proteins bind to and activate a protein serine/threonine kinase such as Raf. Raf stands at the head of a MAP kinase cascade (Fig. 13.32).

D. Downstream members of a MAP kinase cascade may regulate (by phosphorylation) a variety of target proteins. Some of these may be transcription factors that turn on specific genes by binding to regulatory sequences near promoters or in enhancers (Fig. 13.35, donít worry about detailed names of the various proteins involved).

How do we know?

1. The role of protein kinases in signalling. Initial studies that elucidated protein kinase activity in signalling were primarily done by E. Fischer and E. Krebs who worked on the mechanism by which epinephrine activates glycogen breakdown in muscle and liver cells (Fig. 13.20; this is also the pathway in which E. Sutherland earlier discovered the role of cAMP). Subsequently, many other labs worked on this and related pathways, all of which involved phosphorylation at serine or threonine residues. In 1977, Ray Erickson found that the src protein, a cancer-related protein made by Rous sarcoma virus (more about this later), functioned as a kinase and Hunter and Sefton showed that it phosphorylated tyrosine residues (pp. 532-533). Critical to the identification of protein kinases is the use of radioactive ATP in which the g 5' phosphate (the one furthest from the ribose) is 32P. Under the right conditions adding g -labeled ATP to a kinase extract will result in 32P-labeled protein (and release of unlabeled ADP). But which protein is labeled and where? The proteins that get phosphorylated (i.e., the kinase substrate) are usually identified using immunoprecipitation (Fig. 3.32) or gel electrophoresis or both. After the gel is run, all the proteins on the gel can be visualized by staining (usually with a staining reagent) and those that have label by autoradiography (Fig. 3.31, 3.32). Whether they are labeled on tyrosine or serine or threonine is tested by digesting the protein (with proteases or chemicals) and running the amino acid products on 2-dimensional thin layer chromatography/electrophoresis plates (p. 532). Each amino acid runs at a particular position, as does each phosphorylated amino acid and the one that was labeled is detected by autoradiography.

Cell signalling can go wrong. As we will soon see, the genes encoding many proteins in cell signalling pathways can mutate during cell outgrowth in a multicellular animal. As one well-studied example, certain mutations in ras genes can cause the protein to get locked in the active (GTP-bound) state. Such mutations aid in establishing uncontrolled cell growth, i.e., cancer. In fact, Ras and many other cell signalling associated proteins were first discovered by their ability to become cancer-causing (oncogenic) and/or the presence of closely related proteins in certain tumor viruses (e.g., the first known protein tyrosine kinase, src, was found in Rous sarcoma virus, a tumor virus discovered in 1911, pp. 532-533 and 614-615.)

 

Food for thought questions, Cell Signalling:

1. The RNA tumor virus, avian erythroblastosis virus (AEV) causes a type of leukemia in chickens. AEV was found to contain two genes not present involved in its ability to cause cancer: erbB was found to be a varient of the epidermal growth factor receptor, and erbA to be a varient of the thyroid hormone receptor. Since all infected cells contain normal genes encoding both of these receptors, what sorts of variations in the viral forms of one or both of these genes (called v-erbA and v-erbB) might lead to the oncogenic (cancer causing) phenotype?

2. Why doesnít protein kinase A directly activate glycogen phosphorylase rather than phosphorylating another enzyme (phosphorylase kinase) that then acts on glycogen phosphorylase?

3. It has been shown to be important that G proteins are attached to the cell membrane (inner side) rather than diffusing freely through the cytoplasm. Why might this be?

4. G protein-coupled receptors trigger a change in their cytoplasmic domain by a conformational change upon transmitter binding, whereas receptor tyrosine kinases activate their cytoplasmic domains by dimerization? Propose a reason why the receptor tyrosine kinases donít just use a conformational change like the G protein-coupled receptors.

Some example transmitters:

Transmitter

chemistry

membrane permeable?

E/P/A

Receptor

example function 

Steroid Hormones

lipid

Yes

E/P--A?

steroid hormone receptor

transcription regulation-cell differentiation

NO

gas

Yes

P

enzyme

blood vessel dilation

Neurotransmitters

hydrophilic small molecules

No

E/P

gated ion channels, G-prot. receptors

neuromuscular synapses, hormones like adrenalin

Peptide Hormones, Neuropeptides, Growth Factors.

peptides

No

E/P/A

varied cell surface receptors

cell growth and differentiation (includes oncogenes)

 

Example Receptors

Receptor

membrane spanning helices

Associated proteins

binding triggers:

downstream results

off mechanism

example

Ion channel

variable

 

channel opens (or closes

membrane depolarization

depolarization closes channel

acetylcholine receptor

G-protein coupled

seven

G protein

Ga b g -GDP® Ga -GTP + Gb g

enzymes (e.g., adenyl cyclase) or channels

Ga -GTP® Ga -GDP

epinephrine receptor  

Receptor tyrosine kinases

1 per subunit

SH2-binding domain proteins

dimerization, auto-tyr P-ltn, target tyr P-ltn

often, protein kinase cascade

tyrosine phos-phatases

epidermal growth factor receptor

Cytokine receptors

1 per subunit

Non-receptor tyr kinase

dimerization, kinase auto-P-ltn, target tyr P-ltn

often, protein kinase cascade

tyrosine phos-phatases

Interferon receptors