C2006/F2402 '06 OUTLINE OF LECTURE #13
Dr. Deborah Mowshowitz, Columbia University, New York, NY.
03/01/2006 11:50 AM
Note: Exam #2 includes topic I, but not the rest of this lecture.
Handouts 13A & 13B. Not on web. You will also need 12A from last time.
I. Signaling with G proteins & Second Messengers, cont. -- How do you get different responses in different cells?
A. The same 2nd messenger can generate different responses (in different cells) For example, epinephrine response in skeletal muscle vs response in smooth muscle of lungs using cAMP. (More details of responses in last lecture.)
1. Receptors are same. Epinephrine receptors in skeletal muscle and smooth muscle of lungs are the same -- activate same G protein and generate the same second messenger (cAMP).
2. Response to same 2nd messenger is different. Response in skeletal muscle is breakdown of glycogen; response in smooth muscle is change in Ca++ (leading to relaxation of muscle).
3. How do you get different responses? In both cases generate cAMP & stimulate PKA, but target proteins (the ones available to be phosphorylated by PKA) are different. Since different proteins are present (in the two types of muscle), different proteins are activated (& inhibited) and you get a different response.
If you haven't done them yet, try problems
6-6 to 6-8.
Also try problem 6-10.
B. A hormone can generate different second messengers (in different cells) For example, effects of epinephrine (adrenaline) on different smooth muscles -- some relax and some contract in response to epinephrine. (More details of responses in last lecture.)
1. Multiple receptors: Many hormones have more than one type of receptor. Different receptors are found in different cell types. In this example, smooth muscles around bronchioles have adrenergic beta receptors; smooth muscles around some arterioles have adrenergic alpha receptors. The different receptors activate different G proteins. (See table in last lecture.)
2. Different Second Messengers: Different G proteins can generate different second messengers; these activate (or inhibit) different target proteins and produce different results in the target tissues.
3. How it works in this example: Two types of adrenergic (epinephrine) receptors activate different G proteins and generate different second messengers as on handout 12A.
(1). Beta receptors → G protein type (Gs)→ cAMP response → PKA → phosphorylation of Ca++ pump → removal of cell Ca++ → relaxation
(2). Alpha1 receptors → different G protein (Gp) → different second messenger (IP3) → Ca++ release from ER → contraction. How is IP3 generated? See below.
C. Details for DAG/IP3/Ca++ pathway. See handout 12A & Becker figs. 14-9 & 14-10 (10-8 & 10-9) or Purves 15.11 (15.13)
→ →→ synthesis of NO (nitric oxide; acts as diffusible signal) → dilation of blood vessels; Viagra prolongs the response to NO. If you are interested, see Becker fig. 14-16 (10-16) or Purves 15.13 (15.15) and accompanying text. (Note: the two texts differ in some of the early steps in the pathway leading to activation of NO synthase. Different signals may activate NO synthase through different pathways.)
1. How IP3 and DAG are generated.
a. Activated G protein binds to phospholipase C (PLC)
b. PLC cleaves PIP2 in membrane --> PIP3 (soluble, in cytoplasm; also known as InsP3) and DAG ( remains in membrane). For structure see handout 12A and Becker fig. 14-9(10-8).
c. Other inositol derivatives are involved in signaling -- this is a current hot subject of investigation.
2. Role of IP3/Ca++
a. IP3 opens Ca++ channels in the ER, raising Ca++ in cytoplasm. (Becker fig. 14-11 (10-10) for IP3 effect; fig 10-11 for overall Ca++ regulation.)
b. Ca++ acts alone or binds to calmodulin; complex (calmodulin-Ca++) or Ca++ alone alters activity of many proteins. (See Becker fig. 14-13 (10-12) for pictures of calmodulin.)
c. Ca++ affects many processes -- sometimes called "3rd messenger." Changes in [Ca++] can trigger exocytosis (& secretion) or muscle contraction & big changes in Ca++ levels are involved in egg fertilization (see Becker fig.14-14 (10-13) or Purves 15.12 (15.14) for some nice pictures).
d. Examples of use of IP3/Ca++ signaling:
(2). In some smooth muscle tissue, epinephrine → receptor → G prot → → IP3 → Ca++ release → contraction.
3. Role of DAG
a. DAG (in membrane) activates protein kinase C (= PKC, not to be confused with PLC).
b. "PKC" is a family of related enzymes involved in many different processes. If you are interested, see Becker or advanced texts for details. Some PKC's require Ca++. PKC and PKA have different target proteins.
Try problems 6-5, 6-9 & 6-16.
II. How do Tyrosine Kinase Linked Receptors Work?
A. Review: What are the 4 basic types of receptors?
1. Steroid Receptors -- Internal (intracellular) receptors for lipid soluble hormones (steroids & TH)
2. GPLRs (or GPCRs) -- Cell surface (transmembrane) receptors linked to G proteins (also called G Protein Coupled Receptors or GPCRs)
3. RTKs -- Cell surface (transmembrane) receptors that are -- or are linked to -- tyrosine kinases.
4. Channels -- some transmembrane receptors are ion channels. These will be ignored for now but discussed at length when we get to nerves & muscles. The most famous example is the nicotinic acetyl choline receptor -- the receptor involved in nerve-muscle signaling.
B. How do GPLR's and RTK's Compare? -- See handout 13A for summary of basic features of TK or TK linked receptors and comparison to G protein linked receptors. See Purves 15.6 (15.7) for structure & mechanism or Becker fig. 14-17 (10-17) for structure and Becker 14-18 (10-18) for mechanism.
C. Summary of Properties of Receptor TKs (See handout 13A).
1. Receptor is usually a single pass protein
2. Ligand binding usually leads to dimerization of receptors (Purves 15.6 (15.7) or Becker 14-17 (10-17).)
3 Dimerization activates a TK in cytoplasmic domain of receptor or in separate TK (that binds to receptor).
4. Active kinase auto-phosphorylates itself. TK adds phosphates (from ATP) to its own tyrosines -- each subunit phosphorylates the others.
5. Other proteins are activated by binding to phosphorylated TK's. (Becker 14-18 (10-18))
a. Some proteins can be activated by binding directly to TK
b. Some proteins are activated by binding indirectly -- these proteins bind to "adapter proteins" that are bound to the TK. How it happens:
(1). Adapter proteins bind directly to the TK
(2). Additional proteins bind to the adapters
(3). Binding to adapters --> activation of the additional "recruited" proteins.
c. Proteins that bind directly to TK have certain types of domains -- usually called SH2 binding domains.
d. Recruitment. Note that the target protein to be activated comes to or is "recruited by" the TK; this is the opposite of the situation with most 2nd messengers where the messenger diffuses throughout the cytoplasm and "seeks out" the target protein to be activated. The recruitment method may be important in localizing the response to a particular part of the cell.
6. What ligands use TK receptors?
Many growth factors (such as EGF) and other paracrines, autocrines and juxtacrines act through TK receptors. Insulin, but not most other endocrines, acts through TK receptors. (See Purves 15.6 (15.7))
7. Activation of TK receptors often leads to changes in TF's and transcription. (Note this not the usual case with G-linked receptors -- their end target is usually not a TF.)
Try problems 6-1 & 6-3
D. Important examples of proteins activated by binding to receptor TK's (directly or through adapters). See handout 13B.
→ IP3 → trigger cascade using IP3. This means that the IP3 pathway can be activated by both types of receptors -- GPCRs and TKs. The cAMP pathway (as far as we know) can only be activated by GPCRs.
1. PLC. Ligand binding to TK-linked receptor can activate a type of PLC
2. ras -- a famous (infamous?) protein. See Purves fig. 15.9 (15.11) or Becker 14-17 (10-18) and handout.
→ binding of adapter proteins → binding of ras to adapter proteins. Binding of ras to the final adapter protein (also called GEF = guanine-nucleotide exchange factor) catalyzes activation of ras = conversion of ras-GDP to ras-GTP.
a. What is ras? It's a small G protein introduced last time.
(1). What makes it a G protein? It has GTP bound (active) and GDP bound (inactive) forms.
(2). What makes it "small?" It's not trimeric -- it has no subunits
(3). Ras (& other small G proteins) require other proteins to catalyze activation (binding of GTP) and to control rate of inactivation (GTP hydrolysis).
b. Why does anyone care about ras?
(1). Overactive form of ras (stuck in the activated 'on' form) can cause cancer.
(2). Large % (about 30%) of tumors have overactive ras.
c. How is ras activated? Activation of receptor TK
d. What does (normal) activated ras do?
(1). Ras works by activating a series (cascade) of 3 protein kinases generally called MAP kinases.
(a). End result of cascade = activation of final kinase in cascade = MAP kinase or mitogen-activated protein kinase. See Purves 15.9 (15.11) or Becker 19-41 (17-39), or handout. Why bother? Achieves high degree of amplification of signal.
(b). Final kinase (MAP kinase) activates TF's → transcription of genes → mRNA → new proteins.
(2). Many growth factors use ras signaling to trigger the events needed for the G1 to S transition. (What new protein must be made to enter S??) See handout for how GF & ras fit in.
(3). 'Ras' is really a family of proteins whose members serve many different important functions in addition to that described above.
e. How does overactive ras cause cancer? Allows improper G1 to S transition.
3. The same signal (same activated TK receptor) may trigger more than one signaling pathway. For example, EGF can trigger both the IP3 pathway and the ras pathway. (Becker fig. 14-18 (10-18)).
E. A human example of TK receptor signaling: FGF (Fibroblast growth factor) and FGF Receptor. See Becker figs. 14-19 & 14-20 (10-19 & 10-20). Important as an example of a "dominant negative."
1. Signaling system uses a TK receptor
2. FGF & FGFR needed for proper development as described in Becker chap.10. Failure of signal transmission causes developmental abnormalities. See Becker fig. 14-20 (10-20).
3. Why is achondroplasia (type of dwarfism), due to a defective FGFR, dominant? See Becker fig. 14-19 (10-19), and box on handout 13A.
a. The general principle: TK receptors must dimerize in order to act. If 1/2 the receptors are abnormal, most of the dimers that form do not work.
b. The consequences: "lack of function" mutations in receptors are often dominant. (For an example see Becker figs. 14-19 & 14-20 (10-19 & 10-20). Dimers form, but they are inactive.
c. This case: In a heterozygote for achondroplasia, 1/2 the FGF receptors are defective. In the defective receptors, the cytoplasmic domain of the protein is missing. Dimers form, but most dimers are never activated -- the two monomers can not phosphorylate each other. Therefore the signaling (required to form bone) is badly disrupted.
4. Dominant Negatives.
a. Definition: An abnormal allele like the one that causes achondroplasia is called a "dominant negative mutation." A dominant negative allele (or mutation) makes an inactive protein that disrupts function even in the presence of a normal allele (and normal protein).
b. 'Dominant negative' means that the heterozygote is negative for function, not that it doesn't produce any protein. There is actually a mixture of normal and abnormal protein present. What's unusual is that the abnormal, inactive, protein 'gets in the way' and interferes with the working of the normal, active, protein. So overall, there is a lack of function in the heterozygote.
c. Negative mutations (those that produce inactive protein) are usually NOT dominant. Most negative or "lack of function" alleles (or mutations) are recessive. If there is a mixture of normal and abnormal protein in the heterozygote, the normal, active, protein usually works (in spite of the presence of abnormal, inactive, protein). So usually, overall, there is NO lack of function in the heterozygote.
d. Significance: What does the existence of a dominant negative mutation imply? It indicates that the gene involved codes for a protein that must polymerize in order to act.
F. Signaling Pathways all interrelate
1. Different 2nd messengers can influence the same enzyme/pathway
2. Each signaling system can affect the others -- For example, Ca++ levels can affect kinases/phosphatases and phosphorylations can affect Ca++ pumps (& therefore Ca++ levels). See an advanced text if you are interested in the details.
To sum up signaling, try problems 6-15, 6-17 & 6-18.
III. Growth Factors, Growth Control, & Cancer -- Putting it All Together.
This material is covered in Becker, chap 24 ( Ch17 p. 562 to end), and Purves Chap 17 pp350-355. We'll finish it next time.
A. What is wrong with cancer cells? Usually G1-S switch is defective. Cycle is normal length (not shorter), but cell does not pause normally at checkpoint -- proceeds too readily from G1 to S.
1. Problem can be at many different steps-- with production of CDK/cyclin complex, with its activation, or with it's targets. (See handout 13B)
2. Two major types of mutations responsible -- result in either
a. Stuck accelerator (stimulation uncontrolled)
b. Brake failure (inhibition fails)
3. Types of mutations that cause cancer used to unravel normal G1/S signaling pathways & vice versa
B. Overview of how mutations mess up G1/S switch signaling
1. Can over-activate an activating (turn on) gene = "stuck accelerator" type of alteration -- activator is always 'on.'
a. Examples: Changes in genes for ras (so it's always in active form), or GF (always produced as autocrine), or GF receptor (always activated even without GF present), etc.
(1). Normal activator gene is called a proto-oncogene
(2). Over-active version is called an oncogene. (See Becker table 24-1 (17-2) for a list)
c. Oncogene can "over do it" 2 ways (see Becker fig. 24-15)
(1). Gene can be overexpressed -- problem is with gene expression = level of mRNA production and/or translation = level of protein synthesis. Protein is normal, but you make too much of it (or you make it at the wrong time/place). Mutation is in regulatory sequence controlling gene, not in coding part. (ex: constitutive production of a GF)
(2). Gene can be altered so protein made is constitutively active -- problem is with regulation of protein (not gene) activity -- protein is altered so it is always active. Mutation is in protein coding part of gene. Normal amount of protein is made, but protein is altered. (ex: production of altered GF receptor that is active without its ligand.)
2. Can inactive a blocking gene = "brake failure" type of alteration -- inhibitor is always 'off.'
a. Terminology; Normal "brake" genes are called tumor suppressors (see Purves 17.17 (18.17) for picture or Becker table 24-2 (17-3) for a list)
b. Examples: Most famous examples of tumor suppressors are rb and p53. (More details next time.)
(1). p53 -- protein that causes block in cell cycle in damaged cells. Normal p53 acts as 'brake' on activation of CDK/cyclin complex . (see Becker fig. 19-39 (17-40))
(2b). rb -- one of the targets of start kinase. rb protein must be phosphorylated (and inactivated) for cell to enter S. Rb 'holds up' the cycle until CDK/cyclin complex is properly activated. (See handout and Becker fig. 19-38 (17-36)).
c. How mutation causes cancer: When p53 or rb (or other brake protein) is defective, cell keeps on growing when it should not -- in spite of damage (p53) or lack of growth factor signal (rb), etc. Result is often a tumor.
3. Types of mutations that cause cancer are used to unravel normal G1/S signaling pathways & vice versa
Try problems 15-1, 15-9 & 15-11.
C. How do Cancer Causing Mutations occur?
1. Somatic mutations can cause cancer (see Becker fig. 24-12)
a. What's a somatic mutation? A mistake in DNA replication that occurs in somatic cells, not in germ cells. Is not inherited or passed on.
b. Changes in Regulation. Genes can become over-active [or inactive] because of rearrangements or other mutations that alter effects of enhancers, silencers, etc. Example of cancer caused by rearrangements of regulatory sequences: Burkitt's lymphoma. (Proto-oncogene is placed next to a very strong enhancer.) See Becker fig. 24-13.
c. Changes in Protein Structure. Protein can become over-active [or inactive] because of changes in the gene that alter the amino acid sequence of the protein. Example caused by rearrangement: chronic myelogenous leukemia (CML -- too many white blood cells) -- cells make a hybrid, constitutively active TK instead of normal TK whose activity is regulated. See Becker Fig. 24-14 & 24-15.
2. Viruses can cause cancer -- viral DNA integrates into normal cell DNA and brings in new genes (or messes up old ones of host).
a. Virus can carry in a new gene/protein -- an oncogene or a gene that codes for the inhibitor of a tumor suppressor. See Becker fig. 24-18.
b. Virus DNA can destroy a host gene -- virus can integrate into DNA in middle of a normal gene and inactivate that gene -- can knock out a tumor suppressor.
c. Virus DNA can carry in a new regulatory sequence -- virus can integrate into DNA near a normal host gene and provide an enhancer or silencer that changes expression of that host gene -- can turn on a proto-oncogene.
d. Most cancers are not caused by viruses. A few types of cancer are associated with viruses (see Purves Table 17.1 (18.2)) but even in these cases the viruses alone are usually not sufficient to cause the cancer. An example: Cervical cancer is largely viral in origin, in that HPV infection is usually involved. However HPV infection is not sufficient to cause it. HPV makes a protein that inactivates rb protein. (Note: there is some recent evidence that some cases of prostate cancer are associated with a viral infection. See also medpage for a reference to the original report.)
3. You can inherit a predisposition to cancer, not the disease itself. How can you inherit a "predisposition" = high chance of getting cancer?
a. Genes that affect DNA replication &/or repair. If you inherit versions of genes giving low repair or high mutability, tend to get cancer -- sooner or later, one of the random mutations that occurs is likely to mess up a proto-oncogene or tumor suppressor gene as above. Example: xeroderma pigmentosum, which carries a high risk of skin cancer because of defects in the genes for DNA repair. See Becker, box 24A & fig. 24A-1.
b. Tumor suppressor mutations. If you inherit one defective copy of a tumor suppressor gene, nothing happens unless the second copy of the gene gets messed up in a cell. If both copies of a tumor suppressor gene in one cell get inactivated or lost, cancer can result. (This is the "two-hit" hypothesis for how mutated tumor suppressors cause cancer. See Purves 17.16 (18.16) or Becker fig. 24-17. Example: retinoblastoma, which causes a high risk of eye and ovarian tumors, is caused by defects in rb.
4. Most cancer is sporadic, due to somatic mutation. Not inherited.
5. Cancer develops in stages
a. Most cancers have more than one mutation
b. Selection -- selection for increasing loss of growth control occurs as disease progresses -- cells that grow more aggressively (due to additional mutations) outgrow the others.
c. Progression: Normal cell --> benign tumor --> malignant (invasive) --> metastasis (spreads) See Purves 17.18 (18.18) or Becker fig. 24-10.
d. What sort of mutation causes cancer? Is cancer caused by a 'lack of function' mutation or 'gain of function' mutation? Usually both.
Try problems 15-3 & 15-4.
Next Time: Wrap up of Growth Control, & Cancer -- Putting it All Together. Then intro to endocrinology.