C2006/F2402 '07 OUTLINE OF LECTURE #11
(c) 2007 Dr. Deborah Mowshowitz, Columbia University, New York, NY. Last update 02/21/2007 09:47 AM .
Handouts: Handouts are 11A (Big Bang Model of Signaling), 11B (Types of signal molecules)
Links to diagrams and animations on signaling
These links are from previous years but should still be useful. If you find other good ones, let me know.
I. Alternative Splicing -- see notes of last time and handout 10B.
To review regulation & alternative splicing, try problems 4-12 & 4-13, & 4R-6.
1I. Regulation at translation.
A. How to control rate of translation? In principle:
1. Can regulate half life of mRNA (control rate of degradation). In prokaryotes most mRNA's have a short 1/2 life; in eukaryotes this is not necessarily so. Different mRNA's have very different half lives. (Note: proteasomes degrade only proteins NOT RNA's. )
2. Can regulate rate of initiation of translation (control how effectively translation starts).
B. Some Famous Example of Regulation of Translation. (The principles are important; we will not go into the details.)
1. Use of a regulatory protein: -- Have a protein that binds to mRNA and affects either initiation and/or degradation, depending on where it binds. Regulatory protein acts like a repressor, but binds to regulatory sequence in mRNA, not DNA. Regulatory protein is allosteric, and level of small molecule effector (Fe, heme, etc.) inactivates the regulatory protein, either directly or indirectly. Two examples:
a. Regulation of synthesis of Ferritin & Transferrin Receptor (& intracellular iron levels). Regulatory protein is inactivated by Fe; Active form of protein binds to some mRNA's at 5'end (blocking initiation) and to some mRNA's at 3' end (blocking degradation). See Becker, figs. 23-33 & 23-34 (21-33 & 21-34) if you are curious about the details.
b. Regulation of globin synthesis by heme (fig. 23-32). Heme (the prosthetic group of hemoglobin) stimulates synthesis of globin (the protein part of hemoglobin). In this case, heme (indirectly) inhibits an inhibitor of translation. In the absence of heme the inhibitor is active, and translation is blocked. In the presence of heme, the inhibitor is inactivated, and translation proceeds. Interesting features of this case worth noting are:
(1). Inhibition of an inhibitor results in stimulation; in other words, (-) X (-) = (+).
(2). The activity of the inhibitor is controlled by phosphorylation. This is another example of a protein that has active and inactive forms, and phosphorylation (or dephosphorylation) interconverts the two forms. (See III A below.)
→ Formation of double stranded RNA. This triggers degradation &/or inhibition of translation of the mRNA, and may also inhibit transcription of corresponding gene.
2. Use of a regulatory RNA -- RNA interference (RNAi)
a. Trans acting factors can be RNA. Not all regulatory factors are protein -- some are short RNA's. (These are usually derived from double stranded RNA -- See Becker figs. 23-35 & 23-36.)
b. How does a short RNA affect translation? Small RNA binds to mRNA
c. Use in Regulation: Cells naturally produce micro-RNA's that bind to mRNA's and block translation as above. The use of short regulatory RNA's to block translation appears to be important during regulation of development. (See Becker 23-36.)
d. Use in the Lab as a tool: Called RNAi = RNA interference. The use
of artificially added short double stranded (ds) RNA to block
transcription/translation and turn genes off is very common. (See Becker
23-35.) Enzymes of cell convert added ds RNA into short single stranded
RNA that interferes with translation and/or transcription as in b. Same
effect as adding antisense RNA (but works better).
III. Post Translational Regulation. Don't forget: regulation occurs after translation too -- after proteins are made, their activity can be modulated. Many examples of post translational modification have already come up and more will be discussed below.
A. Covalent Modification. Proteins can be modified covalently either reversibly (for ex. by phosphorylation and dephosphorylation), or permanently (for ex. by removal of N-terminal met., addition of sugars -- glycosylation, etc.)
See problem 6-3.
B. Noncovalent Modification. Proteins can be activated or inhibited by reversible noncovalent binding of other factors -- small molecule allosteric effectors, other proteins such as calmodulin (an important Ca++ binding protein to be discussed later), etc.
C. Degradation. Proteins can be selectively destroyed.
1. Half Lives Vary. Not all proteins have the same half life.
2. Proteasome: Major factor in regulation of protein turn over is control of addition of ubiquitin leading to destruction by proteasome. See Becker 23-38 (21-36) or Purves 14.22 (14.21) or the Nobel Prizes for 2004.
3. Significance: Important example of a family of proteins that all have a short half life = cyclins; control progression through cell cycle. Different cyclins control G1 to S, G2 to M etc. Cyclins are made as needed and degraded immediately after use. (Note: Both mRNA's for cyclins and cyclins themselves are degraded after use. More on this when we cover the details of the cell cycle.)
D. Location. Proteins can activated or inhibited by a change of location. For example, transporters like GLUT4 only work if positioned in the plasma membrane; if they are sequestered in vesicles they are inactive. Transport of glucose into the cell can be regulated by moving the GLUT4 in and out of the membrane.
To review post-transcriptional &/or post-translational regulation, try problem 4-14. By now you should be able to do all the problems in 4 & 4R except for a few fine points about hormones, which should be covered below.
IV. Introduction to Signaling -- How are messages sent from one cell to another? How are events in a multicellular organism coordinated? It's not enough to regulate what one cell does!
A. Usual Method -- one cell secretes a signal molecule that binds to a receptor inside -- or on the surface of -- the target cell→ amplification → big effect.
B. How do secreted signal molecules work at molecular level? Overview. See handout 11A
1. Signals are evolutionarily conserved. Same signal molecules used by different organisms for different purposes.
2. Role of Receptors. First step in signaling is binding (noncovalent) of signal to receptor, causing conformational change in receptor.
3. Amplification or the Biological Big Bang. All signals → amplification = big effect from a small concentration of signal. Example: 1 molecule of epinephrine can cause release of 108 molecules of glucose from a liver cell ! (See Becker 14-3 for the calculation.)
4. How is amplification achieved? How is the 'Big Bang' Accomplished? Three ways:
a. By affecting transcription/translation → lots of new protein made → big effect
|ligand binds||→||activate a TF||→||transcribe a gene||→||make lots of mRNA molecules/gene||→||mRNA translated||→||many new protein molecules/mRNA|
Example: Thyroid hormone (thyrotropin or TH) & steroid hormones. Receptor is itself a TF.
b. By cascades of modification → lots of (pre-existing) protein is modified→ big effect. General idea:
|ligand (1st messenger) binds||→||activate receptor in membrane||→||activate protein inside cell (usually a chain of activations)||→||activate target protein (enzyme, or TF, etc.)||→||lots of product|
Specific Example: Thyroid stimulating hormone (TSH) -- promotes release of thyroid hormone (TH).
|ligand (1st messenger) binds||→||activate receptor in membrane||→||activate G protein in the membrane||→||activate enzyme in membrane||→||generate small molecule (2nd messenger) inside cell|
Details of G proteins, second messengers, etc. will be discussed later. In this case 2nd messenger is cAMP, and remaining steps are:
|cAMP||→||activate protein kinase (PKA) in cytoplasm||→||phosphorylate target enzymes||→||stimulate multiple steps in synthesis and release of TH|
Almost every step in this process involves an amplification -- each protein modifies multiple target molecules or generates multiple molecules of product. For an estimate of the amount of amplification involved, see Becker 14-3 (target enzymes in Becker example are different, but rest is the same)*.
* The usual example for this type of modification cascade is breakdown of
glycogen, stimulated by epinephrine (adrenaline), which is the example in Becker
fig. 14-3. If you are interested in more details of the pathway, see
Purves 15.15 (15.17. ) or Becker fig. 14-24. (This example was the first to be
discovered, but is more complex. It will be discussed later.)
c. By opening (ligand-gated) channels
|ligand binds||→||open a few ligand gated channels||→||a little ion flow||→||hit threshold voltage||→||open many (voltage-gated) channels||→||big change in ion concentrations||→||big effect|
Example: Acetyl choline (AcCh). AcCh receptor is a Na+ channel opened by AcCh.
4. Types of Signals. Two main kinds of chemical signals -- lipid soluble and water soluble
|Signal Type||Example||Receptor Type||Effect|
|Lipid Soluble||Thyroxine, steroids||Intracellular*||Gene activity|
|Water Soluble||Peptide hormones, GF's||Cell Surface||Protein activity (usually)|
*Note: Some lipid soluble signals have cell surface receptors in addition to their intracellular receptors. One such case is in the problem book. Cell surface receptors for lipid soluble signals have been discovered relatively recently, and will largely be ignored in this course.
5. Types of Receptors -- intracellular and on cell surface See Purves 15.4 (15.5)
a. Intracellular -- for lipid soluble signals. All similar, all TF's -- details below.
b. On Cell Surface -- for water soluble signals. See Becker fig. 14-2.
(1). Receptors are transmembrane proteins with an extracellular binding domain for signal. These are sometimes called "extracellular receptors" but only ligand binding domain is extracellular, not the entire protein.
(2). Three major kinds of cell surface receptors -- Listed here for reference. Details of structure/function will be discussed as we go.
(a). G Protein Linked Receptors; Also called G Protein Coupled Receptors or GPCRs. (See TSH example above. More details to follow.)
(b). Tyrosine Kinase (TK) Linked Receptors. (These will be discussed later.) These generate cascades of modifications, but do not always use 2nd messengers. If you want to see an example, see Purves 15.9 (15.11).
(c). Ion Channels. Receptor is part of an ion channel. (See AcCh Receptor above). To be discussed at length when we get to nerves. See Purves 15.5 (15.6).
Try problems 6-12 & 6-13.
C. Major types of secreted Signals -- classified by type of cell that makes them and/or target location. See Handout 11B for pictures -- numbers of pictures match numbers below.
→ target (often far away). For an example see Becker fig. 14-22. Insulin, TSH & TH are examples.
1. Endocrine: Endocrine (ductless) gland secretes signal molecule (hormone) into blood
2. Paracrine: Receptor is on adjacent cells. Act locally. See Becker fig. 14-1, p. 414, & table 14-4 for paracrine (or autocrine) vs. endocrine. Examples:
a. histamines (mediate allergic reactions, responses to inflammation)
b. prostaglandins -- initiate uterine cramps; cause fever in response to bacterial infection.
3. Autocrine: Receptor is on same cell. ex. = some growth factors
4. Neurocrine: Neuron secretes neurotransmitter, acts on receptors on neighbor (gland, another neuron or muscle) -- like a paracrine. Example: norephinephrine, acetyl choline.
5. Neuroendocrine: Neuron secretes signal molecule that acts like a hormone (travels through blood to target). Example: Epinephrine (adrenaline).
6. Exocrine: Exocrine gland secretions are released by ducts to outside of body. (Compare to endocrine.) These secretions can carry signals→ target in different individual = pheromones (detected by olfactory receptors in mammals).
D. Other types of Signaling
→ synchronized contractions.
1. Gap Junctions -- allow ions & currents to flow directly from cell to cell -- used in smooth muscle
2. Juxtacrine. Cell surface proteins from two different cells contact -- used in immune system. Similar to basic system, but signal molecule is not secreted -- remains on cell surface.
Good way to study this: Make a
table summarizing C & D above. Include name of type of signaling, source of
signal, type or location of target cell, any other important features, and
an example of each.
V. Properties of Intracellular Receptors (& their lipid soluble ligands). See Purves 15.8 (15.9)
A. All these receptors are similar -- All members of same gene/protein family
B. All these receptors are Transcription Factors
1. Effect on transcription. Some activate and some repress transcription.
2. HRE -- hormone response elements
- All intracellular receptors bind to cis acting regulatory elements upstream of start of transcription.
- Binding site for receptor/TF usually called a hormone response element or HRE.
- HRE's are usually proximal to the core promotor -- can be considered part of promotor, or as separate proximal upstream sites.
C. What sorts of ligands use these receptors?
1. Lipid soluble ligands -- Steroids, thyroxine, retinoids (vitamin A), and vitamin D.
2. Lipid soluble ligands cannot be stored -- must be made from soluble precursors as needed.
D. These receptors have (at least) three domains
1. DNA binding domain -- binds to HRE (different HRE for each dif. hormone)
2. Ligand binding domain -- binds particular steroid (or thyroxine, etc.)
3. Transcription activating (or inhibiting) domain -- also called transactivating domain (for an activator). Binds to other proteins and activates or inhibits transcription.
4. Other domains -- Receptors also need NLS and region that allows dimerization -- these may be separate or included in domains listed above.
E. What (usually) happens when receptors bind their ligands (= receptors are activated?)
1. Receptors disassociate from inhibitory proteins.
2. Receptors dimerize -- form pairs.
3. If receptor is in cytoplasm, moves to nucleus.
- Steroid receptors are generally homodimers; found in cytoplasm (w/o ligand); move to nucleus when ligand binds.
- Other receptors (for vit. D, TH, Retinoic acid) are heterodimers; found in nucleus; activated when ligand binds
4. Receptor (+ ligand) binds DNA if not already bound.
5. Activated receptor binds to other proteins associated with the DNA (other TF's and/or co-activators), and stimulates or inhibits transcription.
F. Example -- Estrogen (A steroid)
1. Basic Mechanism. E → binds to estrogen receptors → complex binds to estrogen response elements (EREs) in regulatory regions of target genes → transcription of some genes activated; transcription of others repressed.
2. Example of some proteins controlled by E -- controls production of receptors for other hormones. For example, during pregnancy controls production of receptors for oxytocin (in uterus) and prolactin (in breast). Oxytocin controls birth contractions; prolactin controls milk production:
a. In uterus: estrogen binding → activates transcription of gene for oxytocin receptors → production of new receptors for oxytocin = up regulation of oxytocin receptors. Receptors needed to allow response to contraction signal (oxytocin) → contractions → birth.
b. In breast: estrogen binding → inhibits transcription of gene for prolactin receptors → down regulation of prolactin receptors; at birth, estrogen level falls and inhibition stops → transcription of gene for prolactin receptors → synthesis of prolactin receptors → response to lactation signal (prolactin).
Summary of Effects of Estrogen
|Affects Receptors for||oxytocin ( → contractions)||prolactin ( → lactation)|
|Effect||Response to oxytocin increases||No response to prolactin|
|Result||Contractions & birth possible||Lactation only after birth (when estrogen levels fall)|
3. Why different results (different patterns of transcription) in different tissues -- in response to the same hormone?
a. May be different hormone receptors in different tissues. Many hormones/signals have multiple types of receptors. (Examples next time.) That's not the explanation here -- in this case, receptors in both cell types are the same.
b. Combinations of TF's (& factors that affect state of chromatin) in each cell are different; often more than one TF is required to get proper transcription of each gene. Hormone signal acts as trigger, but type of hormone effect (what is triggered) depends on other factors.
c. Reminder: All cells have the same DNA. Therefore
- All cells (except immune system) have the same cis acting regulatory sites -- the same HRE's, enhancers, etc.
- It's the trans acting factors & hormone receptors that vary, not the cis acting regulatory sites.
- All cells have the same genes for the trans acting factors, receptors etc., but different genes are used to make regulatory proteins in different cells.
Other examples of how hormones can give different results in different tissues will be discussed next time. In general, what any hormone does depends on combination of proteins (enzymes, TF's, etc.) already in target cell.
Try problem 6-19. By now you should be able to do 6-12 to 6-15.
Next Time: Properties of receptors for water soluble signals; G proteins & second messengers.