C2006/F2402 '07 OUTLINE OF LECTURE #12

(c) 2007 Dr. Deborah Mowshowitz, Columbia University, New York, NY. Last update 02/27/2007 10:03 AM . See sections in blue for corrections & clarifications. (Or see corrections page for a quick summary.)

Handouts:  12A (G proteins & second messengers) & 12 B (How epinephrine controls glycogen breakdown)

I. How do Intracellular Receptors transmit signals from lipid soluble ligands?  See notes of last lecture, topic V.

Try problem 6-19. By now you should be able to do 6-12 to 6-15.

II. 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 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. Binding of activated receptor to G protein triggers activation of G protein.

2. Mechanism of activation  & inactivation

a. Activation (GTP/GDP exchange):

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

b. Inactivation (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. 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 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 to catalyze steps in activation or inactivation. Binding to the activated receptor turns 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 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 steps in activation; for catalysis of inactivation, additional proteins are not required 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 Purves 15.7 (15.8)) or open/close ion channels. More details below.

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

a. Type 1: Linked to G proteins.

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

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

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

(1). Receptor Tyrosine Kinases (RTKs) also called  TK linked receptors

(2). Structure: All are single pass proteins; usually aggregate into dimers when activated.

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. These kinases usually add phosphates to themselves. Ordinary kinases usually add phosphates to other proteins.

Try problems 6-1 & 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?

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 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  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, 6-7, 6-9 A & B.

    C. The same hormone can generate different effects on different tissues  -- How?

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) & table below). 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?

Try problem 6-11.

IV. A Different Second Messenger

    A. Different receptors can activate different G proteins

1. The phenomenon:

a. Epinephrine (secreted in response to stress) has different effects on different smooth muscles:

           (1).  On some smooth muscles, epi contraction
           (2). On other smooth muscles, epi   relaxation (as above)

b. How does this make sense?

(1). In peripheral circulation, smooth muscles around blood vessels contract, diverting blood from peripheral circulation to essential internal organs

(2). In lungs, smooth muscles around bronchioles relax, so lungs can expand more and you can breathe more deeply.

2. How Ca++ fits in:

a. Ca++ stimulates muscle contraction.

b. Epinephrine binds to receptors on some smooth muscles Ca++ released from ER intracellular Ca++ up stimulates contraction. 

c. Epinephrine binds to receptors on some muscles Ca++ pump activated Ca++ removed from cytoplasm intracellular Ca++ down relaxation! In other muscles, activation of receptor reduces response to Ca++ but does not change Ca++ levels.  

3. Role of receptors

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 of receptors activate different G proteins and generate different second messengers as on handout 12A.

(1). Beta receptors G protein type (Gs) cAMP response PKA

In heart muscle, PKA has many effects, including phosphorylation of Ca++ pump removal of cell Ca++ faster relaxation time
Note: Ca++ has many effects on heart, and the net result is to improve pumping, not slow it down.

In smooth muscle, PKA phosphorylates a kinase (MLCK), inactivating it. This prevents any response to Ca++.
For smooth muscle to contract, active MLCK must bind Ca++ . (Ca++ must be in complex with calmodulin; see below.)  If MLCK is phosphorlyated, Ca++/calmodulin cannot bind to it, and contraction does not occur.

(2). Alphareceptors different G protein (Gp) different second messenger (IP3) Ca++ release from ER contraction

4. How does this all work to allow appropriate response  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 (agonists) have been developed that stimulate one and not the other (unlike epinephrine).  Many drugs either imitate or block the effects of signaling molecules such as hormones, transmitters, etc.

Try Problem 6-8 & 6-9C.

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
2nd Messenger IP3 cAMP
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. Details for DAG/IP3/Ca++ pathway. See handout 12A & Becker figs. 14-9 & 14-10 (10-8 & 10-9) or Purves 15.11 (15.13)

1. How IP3 and DAG are generated.

a. Activated G protein binds to phospholipase C (PLC)

b. PLC cleaves PIP2 in membrane PIP3 (soluble, in cytoplasm; also known as InsP3) and DAG ( remains in membrane). For structure see handout 12A and Becker fig. 14-9(10-8).

c. Other inositol derivatives are involved in signaling -- this is a current hot subject of investigation.

2. Role of IP3/Ca++

a. IP3 opens Ca++ channels in the ER, raising Ca++ in cytoplasm. (Becker fig. 14-11 (10-10) for IP3 effect; fig 10-11 for overall Ca++ regulation.)

b. Ca++ acts alone or binds to protein named calmodulin; complex (calmodulin-Ca++) or Ca++ alone alters activity of many proteins. (See Becker fig. 14-13 (10-12)  for pictures of calmodulin.)

c.  Ca++ affects many processes -- sometimes called "3rd messenger." Changes in [Ca++] can trigger exocytosis (& secretion) or muscle contraction & big changes in Ca++ levels are involved in egg fertilization (see Becker fig.14-14 (10-13) or Purves  15.12 (15.14) for some nice pictures). 

d. Examples of use of IP3/Ca++ signaling: 

(1). Contraction of smooth muscle in response to epinephrine as above. Epinephrine (alpha) receptor G prot → → IP3 Ca++ release contraction.

(2). Viagra & NO: IP3 →→ synthesis of NO (nitric oxide; acts as diffusible signal) dilation of blood vessels; Viagra prolongs the response to NO. If you are interested, see Becker fig. 14-16 (10-16)  or Purves 15.13 (15.15) and accompanying text. (Note: the two texts differ in some of the early steps in the pathway leading to activation of NO synthase. Different signals may activate NO synthase through different pathways.)

3. Role of DAG

a. DAG (in membrane) activates protein kinase C (= PKC, not to be confused with PLC).

b. "PKC" is a family of related enzymes involved in many different processes -- act by phosphorylating other proteins. If you are interested, see Becker or advanced texts for details. Some PKC's require Ca++.  PKC and PKA have different target proteins.

Try problems 6-5, 6-10 & 6-16.