C2006/F2402 '04 OUTLINE OF LECTURE #11

(c) 2004 Dr. Deborah Mowshowitz, Columbia University, New York, NY. Last update 02/26/2004 10:14 AM .

Handouts: You need 10C from last time.

A series of links to diagrams and animations on signaling is at http://www.columbia.edu/cu/biology/courses/c2006/links04/signallinks.html. These links are from previous years but should still be useful.

I. Introduction to Signaling -- How are messages sent from one cell to another? How are events in a multicellular organism co-ordinated?

    A. Usual Method -- one cell secretes a signal molecule that binds to a receptor on (or in) the target cell --> amplification --> big effect. 

    B. How do secreted signal molecules work at molecular level? Overview. See top part of handout 10C.

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 of signal to receptor, causing conformational change in receptor.

3. Amplification = Big Bang Theory. All signals --> amplification = big effect from a small concentration of signal. How is amplification achieved? 

a. By affecting transcription/translation --> lots of new protein --> big effect

b. By cascades of modification and/or second messengers (see ** below)  --> lots of modified protein --> big effect. For examples see Purves 15.11 or 15.17.

c. By opening (ligand-gated) channels --> ion flow --> open more (voltage-gated) channels --> big change in ion concentrations --> big effect 

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)

5. Types of Receptors -- intracellular and extracellular. See Purves 15.5

a. Intracellular -- all members of same gene/protein family. All Transcription factors. (Details below.)

b. On Cell Surface -- have Extracellular binding domain for signal -- three main types. (These are sometimes called "extracellular receptors" but only ligand binding domain is extracellular, not the entire protein.)

Receptor Type 2nd Messenger?** Example = Receptor for Usually Affects
G protein Linked Yes (cAMP, IP3 & DAG, or Ca++) Epinephrine & many other hormones Protein Activity
Tyrosine Kinase (TK) Linked Sometimes (IP3) GF's, insulin Gene or Protein Activity 
Ion Channel* No Acetyl Choline (nicotinic) Ion flow

* To be discussed at length when we get to nerves. (See Purves 15.6) The other two types will be discussed below or in next few lectures.
** "First messenger" is the hormone or signal itself. "Second messenger" is small molecule generated inside cell in response to signal.

    C. Major types of secreted Signals -- classified by type of cell that makes them and/or target location. See bottom of Handout 10C for pictures -- numbers of pictures match numbers below..

1. Endocrine: Endocrine gland secretes signal molecule (hormone) into blood ---> target (often far away)

2. Paracrine: Receptor is on adjacent cells. Act locally. 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.

5. Neuroendocrine: Neuron secretes signal molecule that acts like a hormone (travels through blood to target).

6. Exocrine: Exocrine gland secretions (exocrine = released outside the body) can carry signals --> target in different individual = pheromones (detected by olfactory receptors in mammals).

    D. Other types of Signaling

1. Gap Junctions -- allow ions & currents to flow directly from cell to cell -- used in smooth muscle --> synchronized contractions.

 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.

II. Properties of Intracellular Receptors (& their lipid soluble ligands). See Purves 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. All bind to cis acting regulatory elements upstream of start of transcription. Binding site usually called a hormone response element or HRE.

    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. 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.

4. Receptor (+ ligand) binds DNA if not already there. 

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

1. Basic Mechanism. E ----> binds to estrogen receptors ---> complex binds to estrogen response elements 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). :

a. In uterus: estrogen binding --> transcription of gene for oxytocin receptors --> production of new receptors for oxytocin (= up regulation of oxytocin receptors). Receptors needed to allow response to signal (oxytocin) for contraction --> 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 B --> synthesis of prolactin receptors --> response to lactation signal (prolactin).

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 in different tissues?

a. May be different receptors (more than one kind/signal) in different tissues.

b. Combination of TF's in each cell different; more than one TF required to get proper transcription of each gene. In general, what any hormone does depends on combination of proteins (enzymes, TF's, etc.) already in target cell.

Examples of how other hormones give different results in different tissues will be discussed below or next time.

Try problem 6-19 which is not in the problem book but is linked here.

III. Properties of Cell Surface Receptors with Extracellular Binding Domain for Signal -- Two Main Types

How does signaling work when the signals don't enter their target cells?? Need to look at properties of cell surface receptors. Properties of both main types are listed here for reference; today's lecture will concentrate on G-protein linked receptors. (TK linked next time.)

Property of Receptor

G protein Linked

Tyrosine Kinases or TK Linked
Type of transmembrane Protein Multipass (7)# Single pass ##
What happens on ligand binding Change in conformation Usually aggregation (dimers)##

Receptor activates (binds to)

G protein Self (other subunit) or separate TK

Nucleotide role in activation

Replace GDP with GTP Add phosphate from ATP to tyr side chains

Activated TK or G protein subunit* binds to:

Adenyl cyclase or
Phospholipase C or
Ligand gated channels

Many dif. proteins, usually protein with SH2 domains**

Can bind a PLC --> IP3 etc..

Which 2nd messengers used? cAMP or IP3 etc. IP3, etc.,  not cAMP
Receptors for Epinephrine, norepineph., ADH, glucagon Insulin and most GF's (EGF)
Usually modify Enzymes, not TF's Transcription Factors

# See Becker Fig. 10-3. 
## See Becker fig. 10-17 (10-16) or Purves 15.7 
*Either part, alpha or beta + gamma may be activator/inhibitor; G proteins can also be inhibitory
**SH2 = sarc homology 2 domain

IV. How do G protein Linked Receptors Work?

    A. What are G proteins? Properties of G proteins (See Becker fig. 10-4)

1. Catalyze GTP/GDP Exchange (followed by hydrolysis of GTP --> GDP)

a. Activation (exchange):

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

b. Inactivation (hydrolysis):

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

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

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

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

b. On activation --> alpha (with the GTP) separates from other 2.

c. Either part -- alpha, or beta + gamma -- may be the "active" part -- act as activator or inhibitor of target

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

e. Trimeric G proteins (unlike small G proteins) catalyze both GTP/GDP exchange and GTP hydrolysis.

3. Small G proteins have no subunits (example: ras); need other proteins to catalyze the addition/exchange of the GTP. Only reaction they catalyze (by themselves) is hydrolysis of GTP.

4. What do Activated G proteins do? 

a.  Active G proteins can be inhibitory or stimulatory.

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

c. Targets usually generate second messengers (see below) or open/close ion channels (see Purves 15.10).

5. Comparison of Protein Kinases, Trimeric (ordinary) G proteins & Small G proteins

Protein Catalyzes What's added to Target Protein? Target Protein How Inactivated?
Protein Kinase Protein + ATP --> ADP  + protein-P Phosphate Self (Usually separate subunit) or dif. protein Separate Phosphatase removes P
G Protein Exchange & Hydrolysis as above GTP Itself Hydrolyzes GTP to GDP (by itself)
Small G Protein Hydrol. as. above GTP Itself; needs separate prot. for activation Same as above

Try problem 6-2.

    B. How do activated G proteins produce second messengers? (See handout 11A) or Purves 15.8

1.  The usual second messengers -- see handout for structures 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.)

2. General Idea: Active G protein (subunit) --> binds to & activates enzyme in membrane ---> generates second messenger. (See Becker fig. 10-6 or Purves 15.8 & 15.12 (38.13) for cAMP; Becker fig. 10-9 or Purves 15.13 (38-15) for IP3 etc.)

    C.  cAMP as a second messenger -- details

1. How is cAMP made?

a. G protein activates adenyl cyclase (AC)

b. cAMP made from ATP by adenyl cyclase (also called adenylyl cyclase); for structure of cAMP see handout and Becker fig. 10-5 [23-7] or Purves 15.12.

2. What does cAMP do?

a. cAMP binds to and activates protein kinase A = PKA. (Also called cyclic AMP dependent protein kinase = cAPK) 

b. "PKA" is really a family of similar enzymes (with different substrate specificities), and most cell types have one or more. 

c. PKA adds phosphates to other proteins

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

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

(3). End result depends on which kinases and phosphatases are targets (substrates) of PKA and which enzymes are present (to be modified) by PKA or the other kinases/phosphatases (at end of cascade)

3. 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.

4. An example: Regulation of glycogen breakdown and synthesis in response to epinephrine. (See handout  & Purves fig. 15.17 (38-14) or Becker figs. 10-25 (10-24) & 6-18 ). See handout 11B.

a. When hormone present: Hormone activates PKA (protein kinase A or cAMP dependent protein kinase) through pathway explained above:

epinephrine --> receptor --> G prot --> Adenyl cyclase --> cAMP --> PKA.

b. PKA initiates a cascade that activates glycogen phosphorylase and inactivates glycogen synthetase. Therefore, glycogen breaks down --> glucose.

PKA --> activates phosphorylase kinase --> activates phosphorylase --> degrades glycogen

PKA --> inactivates glycogen synthetase

c. When hormone is absent, cAMP is degraded, PKA is not active, and phosphatases reverse effects of PKA. Result is to activate glycogen synthetase and inactivate glycogen phosphorylase. Therefore, glucose polymerized  -->  synthesis of glycogen

Phosphatases  --> activate glycogen synthetase --> synthesis of glycogen  from glucose.

Phosphatases inactivate phosphorylase kinase & phosphorylase

d. Have two controlled processes -- glycogen synthesis and breakdown; system ensures only one works at a time.

Try problems 6-6 to 6-8.

Next time: Details for DAG/IP3/Ca++ pathway; signaling with TK linked receptors