C2006/F2402 '08 OUTLINE OF LECTURE #12

(c) 2008 Dr. Deborah Mowshowitz, Columbia University, New York, NY. Last update 03/03/2008 02:04 PM .
Handouts:    12A  -- G proteins & second messengers
                    12B  -- Modes of Communication between Cells 
                    12C --  Structure/Formation of Glands; Structure of G proteins and GPCR's
                    12D -- How epinephrine controls glycogen breakdown.

I. Introduction to Signaling, cont.

    A. The Story so Far -- Summary of the Big Bang Theory -- see Handout 11A

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

2. Signals -- lipid soluble vs water soluble

3. Amplification -- 3 basic methods

    B. 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. 14-22.

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

d. Structure of gland: For endocrine (ductless) vs exocrine (with duct) see handout 12C.

2. Paracrine: See Becker fig. 14-1,  p. 414, & table 14-4 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. (Compare to endocrine.)  These secretions can carry signals target in different individual = pheromones (detected by olfactory receptors in mammals).

    C. 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 & V 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. 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 (= receptors are activated)

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.

    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 transcription of some genes activated; transcription of others repressed. (Details of mechanism unknown.)

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.

III. How do Transmembrane Receptors (using G proteins) Work?

    A. What is the role of G proteins in signal transduction? Typical Pathway (see also handout 12A):

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

   B. What types of receptors use G proteins? There are two main types of cell surface receptors (other than 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.)

2. Type 2:  Not linked to G proteins. (Will be discussed later when we get to cell cycle and cancer.)

a. Receptor Tyrosine Kinases (RTKs) -- also called  TK linked receptors Have an intracellular kinase domain or interact with an intracellular kinase (when activated).

b. Structure: All are single pass proteins; usually aggregate into dimers when activated.

    C. What are the properties of G proteins? (See Becker fig. 14-5 (10-4))

1. Have active and inactive forms; Active form is bound to GTP; inactive form to GDP.

2. What triggers activation? Binding of activated receptor to G protein triggers activation of G protein.

3. What triggers inactivation? No trigger required -- happens automatically. Therefore G protein does not stay active for long. "Turns itself off."

4. Mechanism of activation  & inactivation

a. Activation Reaction (GTP/GDP exchange):

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."

e. G protein itself has enzymatic activity -- catalyzes both reactions:  activation (exchange) and inactivation (hydrolysis).

f. Binding of activated GPCR (GPCR + signal) turns on enzymatic activity of G protein. 

2. 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

e. Trimeric G proteins catalyze own activation and inactivation. 

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

(2). No additional proteins needed for catalytic steps in activation or inactivation. However binding to the activated receptor needed to turn on the catalytic activity of the trimeric G protein.

3. Small G proteins -- to be discussed further when we get to cell cycle & cancer.

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. Small G proteins can catalyze own inactivation, but not  own activation.

(1). Can catalyze GTP hydrolysis, but not GTP/GDP exchange.

(a). Activation: Small G proteins need other proteins to catalyze the addition/exchange of the GTP --  cannot catalyze exchange of GTP for GDP (and get activated) by themselves.

(b). Inactivation: Small G proteins can inactive themselves by catalyzing hydrolysis of GTP; inactivation often slow.

(2). Additional proteins are required to catalyze activation;  additional proteins are not required for catalysis of inactivation, but are often used to speed up the hydrolysis.

4. What do Activated G proteins do? 

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 G proteins are usually known as Gp, Gq Gi, Gs etc. (Books differ on details of naming.)

e. Targets: G proteins involved in signaling usually activate enzymes that generate second messengers (see Sadava 15.7 or open/close ion channels. More details below.

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

Protein Catalyzes What's added to Target Protein? Primary 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 described above. GTP Itself Hydrolyzes GTP to GDP (by itself)
Small G Protein Hydrolysis of GTP as described above. GTP Itself; needs separate prot. for activation (to catalyze exchange) Same as above

**Receptor protein kinases have an extracellular ligand binding domain and an intracellular kinase 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.

    D. How do activated G proteins produce second messengers? (See handout 12A) or Sadava 15.7 (15.8)

1. General Idea: Active G protein (subunit) binds to & activates enzyme in (or associated with) membrane generates second messenger in cytoplasm. (See Becker fig. 14-7 (10-6) or Sadava 15.7  & 15.12 (15.10) for cAMP pathway; Becker fig. 14-10 (10-9) or Sadava 15.13 (15.11) for IP3 etc.)

2.  The usual second messengers -- see handout 12A 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.)

IV. An example of a second messenger -- cAMP  & its target (PKA) (see handout 12A)

    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 (10-5) or Sadava 15.12 (15.10).

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 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 notes of last lecture. PKA phosphorylates (and activates) enzymes needed to make thyroid hormone.

2. Glycogen metabolism: Consider the specific enzymes involved in regulation of glycogen breakdown and synthesis in response to epinephrine. (See handout  12D & Sadava fig. 15.18 (15.15) or Becker figs. 14-24 (10-25) & 6-17 (6-18 ).

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 into glucose. (For the effectiveness of the cascade, see Becker fig. 14-3.)

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 is polymerized, and there is 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, 6-7, 6-9 A & B. Omit these if no glycogen example!!

    C. The same hormone can generate different effects on different tissues  -- How? If there is no time, this will be discussed after nerves.

1.  Using cAMP in both tissue/cell types

a. An example:

(1). In skeletal muscle: epinephrine causes glycogen breakdown.

(2). In smooth muscle of lung: epinephrine causes muscle relaxation.

b. Why does this make sense?

(1). Epinephrine (also called adrenaline) is produced in response to stress.

(2). In response to stress,  need to "mobilize" glucose -- release it from storage so it can be broken down to provide energy. Therefore need to increase glycogen breakdown (and decrease glycogen synthesis) in muscle (& liver).

(3). In response to stress, need to breathe more deeply. Therefore need smooth muscle around tubes that carry air (bronchioles) to relax.

c. How is this possible?  Same receptors, same 2nd messenger (cAMP) are used. (Solution to be discussed in class. Compare to different effects of estrogen in different target tissues.)

  2. Using different second messengers in different cell types 
(See Becker fig. 14-23 (10-24). An example -- effects of epinephrine (adrenaline) on smooth muscle. Some smooth muscles relax, and some contract in response to epinephrine. In this case, different receptors & 2nd messengers are involved. How does this work? To be continued after nerves.

Try problem 6-11.

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