C2006/F2402 '09 OUTLINE OF LECTURE #12

(c) 2009 Dr. Deborah Mowshowitz, Columbia University, New York, NY. Last update 03/06/2009 09:29 AM .
Handouts:  12A. Overview of Signaling  -- the Biological Big Bang Theory
                  12B  -- Modes of Communication between Cells 
                  12C --   Structure of G proteins and GPCR's; cAMP pathway

Some Interesting/Useful Links
Discovery of G proteins. The 1994 Nobel Prize was awarded to Gilman and Rodbell for discovering G proteins and the processes of signal transduction.  The "Press Release" describes their scientific findings, and there are neat illustrations and biographical notes.  

A hypertext of endocrinology with some animations is at
http://arbl.cvmbs.colostate.edu/hbooks/pathphys/endocrine/index.html

Several animations of Signaling are at the McGraw Hill Web site for the Raven text book.

See the Web-Links page for more links to animations & other online resources

I. 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 signal molecules that bind to a receptor on (or inside) a target cell → amplification →  big effect in target cell. 

    B. How do secreted signal molecules work at molecular level? Overview. See handout 12A

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 opening (ligand-gated) channels

(1). General Idea:

 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  

                (2). Specific example: Acetyl choline (AcCh) effects on muscle. Signaling by AcCh is important in both muscle & nerve responses. AcCh receptor is a Na+ channel in plasma membrane opened by AcCh.

 AcCh binds →  open a few ligand gated Na+ channels →  a little Na+ flows in → cell becomes less - inside; hits threshold voltage → open many (voltage-gated) Na+ channels → big change in Na+ concentrations →   Muscle contracts  

Note: Specific examples are here primarily for reference. More details of each specific example will be discussed below or in later lectures.

b. By cascades of modification  →  lots of (pre-existing) protein is modified→  big effect.

    (1). General idea:

ligand (1st messenger) binds → activate receptor in membrane → activate protein inside cell (usually a chain of activations = cascade*) → activate a lot of target protein (enzyme,  or TF, etc.) → lots of product

            (2). Specific Examples: TSH & epinephrine (2 hormones). TSH stimulates release of thyroid hormone from thyroid gland. Epinephrine stimulates glycogen breakdown. Many water soluble hormones work in this way.  Details of receptors, cascades etc. later.

* The first example for this type of modification cascade to be discovered was the breakdown of glycogen, stimulated by the hormone epinephrine (adrenaline). For details on the extent of amplification, see Becker fig. 14-3 or Sadava 15.18 (15.15).

c. 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 (also called thyrotropin or TH) & steroid hormones. Most lipid soluble hormones act in this way. Receptor is itself a TF.

Question: How does the speed of signaling compare for the three types of amplification?

5. 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.

Question: Which types of signals are usually released by exocytosis?

6. Types of Receptors -- intracellular and on cell surface See Sadava 15.4

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. (See bottom of 12A). Details of structure/function will be discussed as we go.

(a). G Protein Linked Receptors; Also called G Protein Coupled Receptors or GPCRs. (TSH  & epinephrine use these.) More details below.  For an example, see Sadava 15.7)

(b). Protein Kinase (usually 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 Sadava 15.6 & 15.10 (15.6 & 15.9). We won't get to details of how these work for a while.

(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 Sadava 15.5.

Try problems 6-12 & 6-13.

    C. Summary of important issues of signaling to consider (so far) -- See handout 12A.

1. Receptors -- cell surface vs internal (Sadava, fig. 15.4)

2. Signals -- lipid soluble vs water soluble

3. Amplification -- 3 basic methods


II. Major Types of Signaling -- From a Different Point of View -- where do the signals come from, and where do they go?

    A. Major types of secreted Signals -- classified by type of cell that makes them and/or target location. See Handout 12B for pictures -- numbers of pictures match numbers below.

1. Endocrine:

a. Signal molecule secreted by specialized cells in ductless (endocrine) gland

b. Gland secretes signal molecule (hormone) into blood.

c. Target cell is often far away. Acts long range. For an example see Becker fig. 14-23 (14-22).

d. Examples: Insulin, estrogen, TSH (thyroid stimulating hormone) & TH (thyroid hormone)

2. Paracrine: See Becker fig. 14-1 & table 14-4 (6th ed) for paracrine (or autocrine) vs. endocrine.

a. Usually secreted by ordinary cells

b. Target cell is near by -- Receptor is on adjacent cells. Act locally.

c. Examples:

(1). histamines (mediate allergic reactions, responses to inflammation)

(2). prostaglandins -- initiate uterine cramps; cause fever in response to bacterial infection.

(3). Many growth factors (like EGF)

3. Autocrine: Like paracrine, except receptor is on same cell. ex. = some growth factors

4. Neurocrine:

a. Neuron secretes signal molecule.

b. Signal molecule acts as a neurotransmitter (NT)

c. NT acts on receptors on neighbor (gland, another neuron or muscle). Acts locally, like a paracrine.

d. Examples: norephinephrine, acetyl choline.

5. Neuroendocrine:

a. Neuron secretes signal molecule, as in previous case.

b. Signal molecule acts like a hormone (travels through blood to target).

c. Example: Epinephrine (adrenaline).

6. Exocrine: Exocrine gland secretions are released by ducts to outside of body (or to inside of body cavity)*. Secretions on outside can carry signals → target in different individual = pheromones (detected by olfactory receptors in mammals).

*Exocrine secretions (not involved in signaling) can be released to spaces inside of the body that connect with the outside, such as the GI lumen.

    B. Other types of Signaling

1. Gap Junctions -- allow ions & currents to flow directly from cell to cell -- used in smooth muscle → synchronized contractions. Sadava fig. 15.19 (15.16).

 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 B & C 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.
 

III. How do Intracellular Receptors Work? See Sadava 15.8

    A. What sorts of ligands use intracellular receptors? What are the properties of the ligands?

        1. All lipid soluble ligands use intracellular receptors -- Steroids, thyroxine (TH), retinoids (vitamin A), and vitamin D.

        2. Lipid soluble ligands cannot be stored -- must be made from soluble precursors as needed.

3. Hormone binding proteins are needed in blood -- All lipid soluble ligands travel in blood bound to proteins.

    B. All intracellular receptors are Transcription Factors

        1. Effect on transcription. Some activate and some repress transcription.

        2. HRE -- hormone response elements

    C. All these receptors are similar -- All members of same gene/protein family. (Note: Not all TF's are members of the same family, but all hormone receptor TF's are related.)

    D. These receptors have (at least) three domains

1. Transcription activating (or inhibiting) domain -- also called transactivating domain (for an activator). Binds to other proteins and activates or inhibits transcription.

2  DNA binding domain --  binds to HRE (different HRE for each dif. hormone)

3. Ligand binding domain -- binds particular steroid (or thyroxine, etc.)

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  -- how receptors are activated & affect transcription

1. Disassociation -- Receptors disassociate from inhibitory proteins.    

2. Dimerization -- Receptors dimerize -- form pairs.

3. Location -- If receptor is in cytoplasm, moves to nucleus.

4. DNA binding -- Activated Receptor (dimerized & bound to ligand) binds to HRE on DNA.

5. Effect on Transcription -- Activated receptor binds to other proteins associated with the DNA (other TF's and/or co-activators), and stimulates or inhibits transcription.

*Details vary somewhat with different hormones (& corresponding receptors)

    F. Example -- Estrogen (A steroid)

1. Basic Mechanism. E → binds to estrogen receptors → complex; complex binds to estrogen response elements (EREs) in regulatory regions of target genes. Binding → increased transcription of some genes (genes activated); decreased transcription of others (genes 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
Target Organ Uterus Breast
Affects Receptors for oxytocin ( → contractions) prolactin ( → lactation)
Receptors are up-regulated down-regulated
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 lipid soluble hormone?

a. May be different hormone receptors in different tissues. Many hormones/signals have multiple types of receptors. (Examples another 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 later. 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.

IV. Types of Cell Surface (Transmembrane) Receptors (See bottom of 12A)

    A. Channels. Some receptors are themselves (parts of) channels. Note that some channels are receptors, and some channels are opened or closed by separate receptors. Channels will be discussed next time by Dr. Firestein.

    B. Types of Cell Surface receptors that are not channels

1. Type 1: Linked to G proteins.

a. Called G protein linked receptors, or G protein coupled receptors (GPCRs)

b. Structure: All are 7 pass transmembrane proteins with same basic structure; all belong to same protein/gene family. (See Becker 14-4.)

c. Many hormones use GPCRs.

2. Type 2:  Not linked to G proteins. To be discussed later when we get to cell cycle and cancer. For reference:

a. Many are protein kinases. In addition to extracellular, ligand binding domain, have an intracellular kinase domain, or interact with an intracellular kinase (when activated).

b. Most well known type: Receptor Tyrosine Kinases (RTKs) -- also called  TK linked receptors.

c. Structure: Usually are single pass proteins that aggregate into dimers when activated.

d. Many Growth Factors use TK linked receptors or related receptors. (See Becker table 14-3 if you are curious).

 V. How do GPCRs & G Proteins Act in Signaling?

    A. Typical Pathway (see also handout 12C)

ligand (1st messenger) binds outside cell → activate receptor in membrane → activate G protein in the membrane → activate target enzyme in membrane → generate small molecule (2nd messenger) inside cell

 Note that the ligand (1st messenger) binds to the extracellular domain of its receptor. The remaining events are inside the cell.

   B. Roles of G Proteins: G proteins involved in signaling usually

1. Activate enzymes that generate second messengers (see Sadava 15.7) as above, or

2. Open/close ion channels.

    C. Second messengers -- See handout 12C or Sadava 15.7 (15.8)

1. How they are made: Active G protein (subunit) → binds to & activates enzyme in (or associated with) membrane → generates second messenger in cytoplasm. See Becker fig. 14-7 or Sadava 15.7  & 15.12 (15.10) for cAMP pathway. We will get to IP3 pathway later. If you are curious, see Becker fig. 14-10 or Sadava 15.13 (15.11))

2.  The usual second messengers -- see handout 12C for structure of cAMP and mode of action

2nd Messenger Where does it come from? How is it made?
cAMP ATP by action of adenyl cyclase
DAG & IP3 membrane lipid by action of  phospholipase C
Ca++ stored Ca++  in ER (or extracellular) by opening channels (in ER/plasma memb.)

3. What do 2nd messengers do? Bind to and activate (or inactivate) target proteins.

4. A Specific Example: Thyroid stimulating hormone (TSH) -- promotes release of thyroid hormone (TH). 

        a. Generation of 2nd messenger (cAMP)

TSH (1st messenger) binds → activate GPCR in membrane → activate G protein in the membrane → activate enzyme in membrane (adenyl cyclase) → generate small molecule (2nd messenger) inside cell = cAMP

        b. Action of 2nd messenger

cAMP → activate protein kinase (PKA) in cytoplasm →  phosphorylate target enzymes → stimulate multiple steps in synthesis and release of TH

        5. Why bother with all these steps?

            a. Many steps involve amplifications. For example, one molecule of active Adenyl cyclase can generate many molecules of cAMP and one molecule of PKA can phosphorylate many molecules of its target enzymes. For an example of the possibilities of a cascade of amplification, see Becker fig. 14-3 or Sadava 15.18.

            b. The usual example for this type of modification cascade is the breakdown of glycogen, stimulated by the hormone epinephrine (adrenaline), which is the example in Becker fig. 14-3.   If you are interested in more details of the pathway, see Sadava 15.18 (15.15) or Becker fig. 14-25 (14-24). This example was the first to be discovered, but is more complex than the TSH case.

VI. How do G proteins Work?

    A. What are the important properties of G proteins? (See Becker fig. 14-5)

1. Have active and inactive forms

        a.  Active form is bound to GTP

        b.  Inactive form is bound to GDP.

2. G-proteins act as switches in many processes (not just signaling)

        a. G protein is activated by dumping GDP and picking up GTP in response to some signal.

        b. G protein inactivates itself by catalyzing hydrolysis of GTP to GDP.

        c. Therefore G protein does not stay active for long. "Turns itself off."

    B. Activation & Inactivation of G Proteins

1. GTP exchange: Mechanism of activation  & inactivation

a. Activation Reaction (GTP/GDP exchange, NOT phosphorylation of GDP):

Protein-GDP (inactive) + GTP →  Protein-GTP (active) + GDP

b. Inactivation Reaction (hydrolysis of GTP to GDP):

Protein-GTP (active) →  Protein-GDP (inactive) + phosphate.

c. Overall: GTP displaces GDP, activating the G protein; GTP is then hydrolyzed (usually rapidly), returning the G protein to its inactive state. 

d. Terminology. Since the overall result is that GTP is hydrolyzed to GDP, G proteins are sometimes called "GTPases."

2. What triggers activation?

        a. General Case: Binding of a protein called a GEF (guanine-nucleotide exchange factor) causes GDP to fall off, and GTP binds.

        b. In signaling: Activated GPCR = GEF. Binding of activated receptor to G protein triggers activation of G protein (causes loss of GDP).

3. What triggers inactivation?

a. G protein itself has enzymatic activity -- catalyzes inactivation (hydrolysis).

b. No trigger required -- hydrolysis of GTP to GDP happens automatically.

c. Other proteins may increase speed of hydrolysis.  They are called RGS proteins (Regulators of G protein Signaling) or GAP proteins (GTPase Activating Proteins).

    C. Types of G proteins

1. Subunits -- Ordinary G proteins are trimeric = they have 3 subunits.

a. Inactive G prot = heterotrimer of alpha, beta, gamma

b. On activation, alpha subunit (with the GTP) separates from other 2 subunits.

c. Either part -- alpha, or beta + gamma -- may be the effector that actually acts on target -- acts as activator or inhibitor of target protein.

d. Hydrolysis of GTP to GDP causes alpha to reassociate with other subunits → inactive heterotrimer

2. Small G proteins -- to be discussed further when we get to cell cycle & cancer. For reference:

a. Structure: Small G proteins have no subunits.

b. Example: the protein called ras -- important in growth control; many cancer cells have over-active ras.

c. Are activated by GTP/GDP exchange, and inactivated by hydrolysis of GTP to GDP, as above.

d. Are not activated by GPCRs directly (other 'middle man' adapter proteins are involved)

3. How many G proteins? 

a. There are many different G proteins. G proteins are involved in a very large number of cellular processes, not just signaling. (We have ignored their importance until now. See Becker for details & many examples.) 

b.  Active G proteins can be inhibitory or stimulatory.

c. Activated G proteins work by binding to and activating (or inhibiting) other target enzymes/proteins

d. Terminology: The different trimeric G proteins are usually known as Gp, Gq Gi, Gs etc. (Books differ on details of naming.)

    D. For reference: Comparison of Protein Kinases, Receptor Protein Kinases, & Trimeric G proteins

Protein Catalyzes What's added to Target Protein? Who gets the P or GTP? How Inactivated?
Protein Kinase Protein + ATP → ADP  + protein-P Phosphate Usually a dif. protein Separate Phosphatase removes P
Receptor Protein Kinase** Protein + ATP → ADP  + protein-P Phosphate Usually  separate subunit of self Separate Phosphatase removes P
Trimeric
G Protein		 Exchange & Hydrolysis as described above. GTP Itself Hydrolyzes GTP to GDP (by itself)

**Receptor protein kinases have an extracellular ligand binding domain and an intracellular kinase (or kinase binding) domain. Ordinary kinases usually add phosphates to other proteins. Receptor kinases usually add phosphates to themselves. (For an example, see Sadava fig. fig. 15.6)

Try problems 6-1 & 6-2.

VII. An example of a second messenger -- cAMP  & its target (PKA)  See handout 12C, Becker fig. 14-7 or Sadava 15.7  & 15.12 (15.10) for cAMP pathway.

    A.   How is cAMP level regulated? What does it do?

1. How is cAMP made?

a. G protein activates adenyl cyclase (also called adenylyl cyclase or AC)

b. cAMP made from ATP by adenyl cyclase; for structure of cAMP see handout and Becker fig. 14-6 or Sadava 15.12 (15.10).

2. What does cAMP do? See Becker table 14-1 (14-5). 

a. cAMP binds to and activates protein kinase A = PKA. (Also called cyclic AMP dependent protein kinase = cAPK) See Becker fig. 14-8.

b. PKA adds phosphates to other proteins

(1). Phosphorylation by PKA can activate or inhibit target protein (target = substrate of PKA)

(2). PKA action can modify other kinases/phosphatases and start a cascade

(3). End result varies. Depends on which kinases and phosphatases in that tissue are targets (substrates) of PKA and/or the other kinases/phosphatases (at end of cascade). See example below.

2. How does signal system turn off when hormone leaves?

a. G protein doesn't stay activated for long: Activated G protein hydrolyzes its own GTP → GDP (→ inactive G protein).

b. cAMP is short lived -- it's hydrolyzed by phosphodiesterase (PDE)

c. In absence of cAMP, PKA becomes inactivated and phosphatases are active that reverse effects of kinases.

    B. How do hormones work through cAMP?

1. TSH -- see above. PKA phosphorylates (and activates) enzymes needed to make thyroid hormone.

2. Glycogen metabolism:  This case is very complex and will be discussed later. If you want to read ahead, see Sadava fig. 15.18 (15.15) or Becker figs. 14-25 (14-24) & 6-17 (6-18 ).

Next Time: (Dr. Firestein) -- Electrical Signaling -- How you get a Big Bang using channels.