C2006/F2402 '06 OUTLINE OF LECTURE #12
(c) 2006 Dr. Deborah Mowshowitz, Columbia University, New York, NY. Last update 02/27/2006 11:55 AM .
Handouts: 12A (G proteins & second messengers) & 12 B (How epinephrine controls glycogen breakdown)
I. 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. FYI:
- 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)
→ 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.
1. Basic Mechanism. E
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 (more than one kind/signal) in different tissues.
b. Combination of TF's (& factors that affect state of chromatin) in each cell is different; often more than one TF is required to get proper transcription of each gene.
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 TF's, receptors etc., but different ones are made in different cells.
Other examples of how hormones can give different results in different tissues will be discussed below and/or 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.
II. How do Transmembrane Receptors (using G proteins) Work?
A. Basic Idea of Signal Transduction -- Need some process to turn an extracellular signal into an intracellular signal. One common way to do this: using a G protein & a second messenger. Role of G protein is discussed first below and then role of 2nd messenger.
B. What is role of G proteins in signal transduction? A typical case (see handout 12A): Signal binds to receptor on outside of cell (extracellular signal = first messenger)→ activates G protein → activates target enzyme → produces signal inside cell (intracellular signal = 2nd messenger = cAMP in this case) → binds to and activates other enzymes (Protein Kinase A in this case) → activates/inhibits other enzymes, etc. More on 2nd messengers below.
C. What are G proteins? Properties of G proteins (See Becker fig. 14-5 (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 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. 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 "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
a. Structure: Small G proteins have no subunits.
b. An example: the protein called ras -- important in growth control; details will be explained later. Many cancer cells have over-active ras.
c. Activation: Small G proteins need other proteins to catalyze the addition/exchange of the GTP -- cannot exchange GTP for GDP (and get activated) without assistance of other proteins
d. Inactivation: Small G proteins can inactive themselves by catalyzing hydrolysis of GTP. However additional proteins 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 Purves 15.7 (15.8)) or open/close ion channels. More details below and/or next time.
5. Comparison of 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|
|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||Same as above|
Try problem 6-2.
D. How do activated G proteins produce second messengers? (See handout 12A) or Purves 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 Purves 15.7 (15.8) & 15.10 (15.12) for cAMP pathway; Becker fig. 14-10 (10-9) or Purves 15.11 (15.13) 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.)|
III. An example of a second messenger -- cAMP & its target (PKA) (see handout 12A)
A. How is cAMP level regulated? What does it do?
→ GDP (→ inactive G protein).
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. 14-6 (10-5) or Purves 15.10 (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 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
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. Same hormone can have different effects on different tissues using cAMP.
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? In some cases, different receptors or 2nd messengers may be involved; examples of this below and/or next time. However, in this case, 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. How does this work at molecular level -- a specific example: Regulation of glycogen breakdown and synthesis in response to epinephrine. (See handout 12B & Purves fig. 15.15 (15.17) 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 to 6-8, & 6-10.
IV. A Different Second Messenger
A. The same hormone can generate different effects (in
different cells) using different second messengers.
(See Becker fig. 14-23 (10-24) & table below). An example -- effects of epinephrine (adrenaline) on smooth muscle.
→ Ca++ released from ER → intracellular Ca++ up → stimulates contraction.
1. The phenomenon: Epinephrine has different effects on different smooth muscles:
On some smooth muscles → contraction (helps divert blood from peripheral circulation to essential internal organs.)
On other smooth muscles → relaxation (example as above -- allows lungs to breathe more deeply)
2. How Ca++ fits in:
a. Ca++ stimulates muscle contraction.
b. Epinephrine binds to receptors on some smooth muscles
c. Epinephrine binds to receptors on some smooth muscles → Ca++ pump activated → Ca++ removed → intracellular Ca++ down → relaxation!
3. How does epinephrine work two ways on same type of tissue?
a. Two basic types of epinephrine receptors -- called alpha and beta adrenergic receptors (adrenergic = for adrenaline). The two types are distinguished by their relative affinities for epinephrine (adrenaline) and norepinephrine (noradrenaline).
b. Some types of smooth muscle have mostly one type of receptors; some the other. (See note to table below.)
c. Two types 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
4. How 2 receptor types help to respond appropriately to stress (epinephrine).
a. Beta type receptors. Beta receptors are found in lung tissue in smooth muscle surrounding bronchioles. Stress (pop quiz, lion in street, etc.)→ epinephrine → muscles relax → bronchioles dilate → deeper breathing → more oxygen → energy to cope with stress.
b. Alpha type receptors. Alpha receptors are found in smooth muscle surrounding blood vessels of peripheral circulation. Stress → epinephrine → muscles contract → constrict peripheral circulation → direct blood to essential organs for responding to stress (heart, lungs, skeletal muscle).
5. Medical Uses of all this.
Epinephrine can be used during an asthmatic attack to relax bronchi and ease breathing. Overuse of this type of broncho-dilator eases breathing temporarily but masks underlying problem (inflammation of lung tissue) and can have additional serious long term effects (from overstimulation of heart which also has beta receptors). Heart and lungs have slightly different types of beta receptors, so drugs have been developed that stimulate one and not the other (unlike epinephrine).
6. Summary of epinephrine effects on smooth muscle (in lung vs peripheral circulation)
Effects of Epinephrine on Smooth Muscle
|Receptor Type||Alpha1 adrenergic||Beta adrenergic|
|Receptor binds||norepinephrine> epinephrine||epinephrine ≥ norepinephrine|
|G protein activates||PLC (phospholipase C)||adenyl cyclase|
|Effect of 2nd messenger||Ion channel in ER opened||PKA activated → Ca++ pump activated|
|Effect on Ca++||Ca++ released into cyto.||Ca++ removed from cyto.|
|Effect on smooth muscle||Contraction||Relaxation|
|Tissue involved||Peripheral Circulation (arterioles)||Lungs (bronchioles)|
|Final Effect||Blood directed to central organs||Breathing easier|
Note: There are more than two types of epinephrine receptors on smooth muscle cells, so epinephrine may affect smooth muscle in other tissues in other ways. (There are subtypes of alpha and subtypes of beta.)
B. Next time: Details for DAG/IP3/Ca++ pathway.How does a hormone generate a second messenger other than cAMP, such as IP3?