C2006/F2402 '04 OUTLINE OF LECTURE #12

(c) 2004 Dr. Deborah Mowshowitz, Columbia University, New York, NY. Last update 03/01/2004 11:56 AM .

Handouts: Need 11-A & B from last time; 12A (TK receptor signaling )

I. Signaling with G proteins & Second Messengers, Cont.

    A. Review of cAMP pathway & regulation of glycogen metabolism (handouts 11A & 11B)

If you haven't done them yet, try problems 6-6 to 6-8.

    B. Details for DAG/IP3/Ca++ pathway (see handout 11A bottom & Becker figs. 10-8 & 10-9 or Purves 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 11A and Becker fig. 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. 10-10 for IP3 effect; fig 10-11 for overall Ca++ regulation.)

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

c.  Ca++ affects many processes -- can trigger exocytosis (& secretion) or muscle contraction & big changes in Ca++ levels are involved in egg fertilization (see Becker fig.10-13 or Purves 15.14 for some nice pictures). 

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

(1). 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. 10-16 [10-15]  or Purves 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.)

(2). In some smooth muscle tissue, epinephrine -->receptor -->  G prot --> --> IP3 -->Ca++ release --> contraction. More details below.

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. If you are interested, see Becker or advanced texts for details. Some PKC's require Ca++.

Try problems 6-5, 6-9 & 6-16.

    C. The same signal can generate different second messengers & therefore different effects (in different cells). (See Becker fig. 10-24 [10-23] & table below). An example -- effects of epinephrine (adrenaline) on smooth muscle.

1. The phenomenon: Epinephrine has different effects on different smooth muscles:

            On some smooth muscles --> contraction
            On other smooth muscles -->  relaxation

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 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)

b. Some types of smooth muscle have mostly one type of receptors; some the other.

c. Two types activate different G proteins. Therefore

(1). Alpha receptors --> G protein type1 -->IP3 response --> Ca++ release from ER --> contraction

(2). Beta receptors --> G protein type 2 --> cAMP response --> PKA --> phosphorylation of Ca++ pump --> removal of cell Ca++ --> relaxation

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

4. Function of responses to epinephrine -- responses to stress.

a. Beta type receptors. Beta receptors are found in lung tissue in smooth muscle surrounding bronchioles (tubes bringing air to lungs). Stress (pop quiz, lion in street, etc.)--> epinephrine --> muscles relax --> bronchi 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



G protein activates


adenyl cyclase

2nd Messenger



Effect of 2nd messenger

Ion channel in ER opened

PKA activated -->Ca++ pump activated

Effect on Ca++

Ca++ released

Ca++ removed

Effect on smooth muscle



Tissue involved

Peripheral Circulation


Final Effect

Blood directed to central organs

Breathing easier

* 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. 

    D. The same 2nd messenger can generate different responses (in different cells). An Example: consider epinephrine response in skeletal muscle vs response in smooth muscle of lungs.

1. Receptors are same. Epinephrine receptors in skeletal muscle and smooth muscle of lungs are both beta -- activate same G protein -->  same second messenger (cAMP). 

2.  Response to same 2nd messenger is different. Response in skeletal muscle is breakdown of glycogen; response in smooth muscle  is change in Ca++.

3. How do you get different responses?  In both cases generate cAMP & stimulates PKA. Differences could be due to differences in PKA and/or its substrates.

a. PKA's in two cell types may be different in specificity -- there are at least two kinds of PKA.

b. Target proteins available for PKA to phosphorylate are definitely different (in the two types of muscle), so you get a different response. 

Q to think about: In skeletal muscle, what enzyme/protein does PKA phosphorylate? (See handout 11-B.) In smooth muscle what enzyme/protein does PKA phosphorylate? (See table above.)

See problem 6-10.

II. How do Tyrosine Kinase Linked Receptors Work?

    A.  Overview  of Properties -- How do properties of 3 basic types of receptors compare? A compare & contrast for:

1.  Steroid Receptors -- Internal receptors for lipid soluble hormones (steroids & TH)

2. GPLRs (or GPCRs) -- Cell surface receptors linked to G proteins (also called G Protein Coupled Receptors or  GPCRs)

3. RTKs -- Cell surface receptors that are (or are linked to) tyrosine kinases

    C. Table of Properties of Two main types of Cell Surface Receptors  -- See table below (= same as table in last lecture) for summary of basic features of  TK or TK linked receptors and comparison to  G protein linked receptors.  See Purves 15.7 for structure & mechanism or Becker fig. 10-17 [10-16]  for structure and Becker 10-18 [10-17] for mechanism. (In even older editions of Becker, signaling is in chap. 23).

Type of Receptor

Property of Receptor

G protein Linked Receptor

Tyrosine Kinase or TK Linked Receptor (RTK)

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. (if any); not cAMP. Often does not use a 2nd messenger.

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 & 15-4.
*Either part, alpha or beta + gamma may be activator/inhibitor; G proteins can also be inhibitory
**SH2 = sarc homology 2 domain

    D. Summary of Properties of Receptor TKs (See handout 12A).

1. Receptor is usually a single pass protein 

2. Ligand binding usually leads to dimerization of receptors (Purves 15.7 or Becker 10-17 [10-16].) In a heterozygote producing 1/2 normal receptors and 1/2 abnormal receptors, most of the dimers do not work. The consequences of this are that "lack of function" mutations in receptors are often dominant. (For an example see Becker figs. 10-19 & 10-20 [10-18 & 10-19].

3 Dimerization activates a TK in cytoplasmic domain of receptor or in separate TK (that binds to receptor).

4. Active kinase auto-phosphorylates itself. TK adds phosphates (from ATP) to its own tyrosines -- each subunit phosphorylates the others.

5. Other proteins are activated by binding to phosphorylated TK's. (Becker 10-18 [10-17])

a. Some proteins can be activated by binding directly to TK 

b. Some proteins are activated by binding indirectly -- these proteins bind to "adapter proteins" that are bound to the TK. How it happens: 

(1). Adapter proteins bind directly to the TK 

(2). Additional proteins bind to the adapters 

(3). Binding to adapters --> activation of the additional "recruited" proteins.

c. Proteins that bind directly to TK have certain types of domains -- usually called SH2 binding domains. 

d. Recruitment. Note that the target protein to be activated comes to or is "recruited by" the TK; this is the opposite of the situation with most 2nd messengers where the messenger diffuses throughout the cytoplasm and "seeks out" the target protein to be activated. The recruitment method may be important in localizing the response to a particular part of the cell.

6. What ligands use TK receptors? Many growth factors (such as EGF) and other paracrines, autocrines and juxtacrines act through TK receptors. Insulin, but not most other endocrines, acts through TK receptors. (See Purves 15.7)

7. Activation of TK receptors often leads to changes in TF's and transcription. (Note this not the usual case with G-linked receptors -- their end target is usually not a TF.)

Try problems 6-1 & 6-3

    E. Important examples of proteins activated by binding to receptor TK's (directly or through adapters)

1. PLC.  Ligand binding to TK-linked receptor can activate a type of PLC --> IP3 --> trigger cascade using IP3. This means that the IP3 pathway can be activated by both types of receptors -- GPCRs and TKs.  The cAMP pathway (as far as we know) can only be activated by GPCRs.  

2. ras -- a famous (infamous?) protein. See Purves fig. 15.11 or Becker 10-18 [10-17] and handout 12A (more details on B).

a. What is ras? It's a small G protein . 

(1). Ras is a G protein -- it has GTP bound (active) and GDP bound (inactive) forms. 

(2). What makes it "small?" It's not trimeric -- it has no subunits

(3). Ras (& other small G proteins) require other proteins to allow activation (binding of GTP) and to control rate of inactivation (GTP hydrolysis).

b. Why does anyone care about ras?  Large % (about 30%) of tumors have messed up ras (= ras stuck in the activated, GTP bound, form). Therefore we know ras has important role(s) in growth control.

c. How is ras activated? Activation of receptor TK -->binding of adapter proteins --> binding of ras to adapter proteins & activation of  ras --  conversion of ras-GDP to ras-GTP.

d. What does activated ras do?

(1). Ras works by activating the series (cascade) of protein kinases generally called MAP kinases. End result = activation of final kinase in cascade = MAP kinase or mitogen-activated protein kinase. See Purves 15.11 or Becker 17-39 [17-38].

(2). Many growth factors work by triggering the G1 to S transition, using ras signaling as follows:

GF --> binds to TK receptor --> activated ras (see above) --> MAP kinase cascade --> activated TF's --> transcription of genes for cyclin --> mRNA for cyclin  (& CDK if cell was in G0) --> cyclin buildup --> activation of CDK (start kinase) --> cell can pass start checkpoint (enter S)

(3). 'Ras' is really a family of proteins whose members serve many different important functions in addition to that described above.

3. The same signal (same activated TK receptor) may trigger more than one signaling pathway. For example, EGF can trigger both the IP3 pathway and the ras pathway. (Becker fig. 10-18 [10-17]).

    F. A human example of TK receptor signaling:  FGF (Fibroblast growth factor) and FGF Receptor.  See Becker figs. 10-19 & 10-20 [10-18 & 10-19]. Important as an example of a "dominant negative."

1. Signaling system uses a TK receptor  

2. FGF & FGFR needed for proper development as described in Becker chap.10. Failure of signal transmission --> developmental abnormalities. See Becker fig.10-20 [10-19].

3. Why is achondroplasia (type of dwarfism), due to a defective FGFR, dominant? See Becker fig. 10-19 [10-18], and box on handout 12A.

a. TK receptors must dimerize in order to act.

b. If 1/2 the TK receptors are abnormal, dimers tend to be abnormal, even if one of the monomers is normal. (In this case, the cytoplasmic domain of the abnormal receptor is missing, so the two monomers can not phosphorylate each other. The dimer forms but is never activated.)

c. In a heterozygote for achondroplasia, 1/2 the FGF receptors are defective, most dimers are defective (can't activate) and signaling (required to form bone) is badly disrupted.

4. Dominant Negatives.

a. Definition: An abnormal allele like the one that causes achondroplasia is called a "dominant negative mutation." A dominant negative allele (or mutation) makes an inactive protein that disrupts function even in the presence of a normal allele (and normal protein). Note: This is not the usual case --  most negative or "lack of function" alleles (or mutations) are recessive.

b. Significance: Existence of a dominant negative mutation implies that the gene involved codes for a protein that must polymerize in order to act.

    G. Signaling Pathways all interrelate

1. Different 2nd messengers can influence the same enzyme/pathway

2. Each signaling system can affect the others -- For example, Ca++ levels can affect kinases/phosphatases and phosphorylations can affect Ca++ pumps (& therefore Ca++ levels). See an advanced text if you are interested in the details.

To sum up signaling, try problems 6-15, 6-17 & 6-18.

III. Growth Factors, Growth Control, & Cancer -- Putting it All Together.

This material is covered in Becker, chap 17 p. 562 [573] to end. We'll finish it next time.

   A. What is wrong with cancer cells? G1-S switch is defective. Cycle is normal length (not shorter), but cell does not pause normally at checkpoint --  proceeds too readily from G1 to S.

1. Problem can be with production of CDK/cyclin complex or with its activation (see handout 10A; more details next time)

2. Two major types of mutations responsible -- result in either

a. Stuck accelerator (stimulation uncontrolled)

b. Brake failure (inhibition fails)

3. Types of mutations that cause cancer used to unravel normal G1/S signaling pathways & vice versa

     B. Overview of how mutations mess up G1/S switch signaling

1. Can over-activate an activating (turn on) gene = "stuck accelerator" type of alteration

a. Examples: Changes in genes for ras (so it's always in active form), or GF (always produced as autocrine), or GF receptor (always activated even without GF present), etc.

b. Terminology:

(1). Normal activator gene is called a proto-oncogene

(2). Over-active version is called an oncogene. (See Becker table 17-2 for a list)

c. Oncogene can "over do it" 2 ways

(1). Gene can be overexpressed  -- problem is with gene expression = level of mRNA production and/or translation = level of protein synthesis. Protein is normal, but you make too much of it (or you make it at the wrong time/place). Mutation is in regulatory sequence controlling gene, not in coding part. (ex: constitutive production of a GF)

(2). Gene can be altered so protein made is constitutively active -- problem is with regulation of protein (not gene) activity -- protein is altered so it is always active. Mutation is in protein coding part of gene. Normal amount of protein is made, but protein is altered. (ex: production of altered GF receptor that is active without its ligand.)

2. Can inactive a blocking gene = "brake failure" type of alteration

a. Example =  p53 -- protein that causes block in cell cycle in damaged cells. Normal p53 acts as "brake" on activation of CDK/cyclin complex. (see Becker fig. 17-40 -- details next time.)

b. Another example = rb -- protein that prevents cell from entering S unless growth factor signaling activates CDK/cyclin (see Becker fig. 17-36 -- details next time). 

b. When p53 or rb (or other brake protein) is defective, cell keeps on growing when it should not -- in spite of damage (p53) or lack of growth factor signal (rb), etc. Result is often a tumor.

c. Normal "brake" genes are called tumor suppressor (see Becker table 17-3 for a list)

Try problem 15-9.

3. How somatic mutations (mutations in somatic body cells) cause cancer

a. Genes can become over-active [or inactive] because of rearrangements or other mutations that alter effects of enhancers, silencers, etc., or

b. Protein can become over-active [or inactive] because of point mutations in the gene that change the protein.

4. How viruses an cause cancer --  viral DNA integrates into normal cell DNA and brings in new genes (or messes up old ones).

a. Virus can carry an oncogene or inhibitor of a tumor suppressor .

b. Virus DNA can integrate into DNA in middle of a normal gene and inactivate that gene -- can knock out a tumor suppressor.

c. Virus DNA can integrate into DNA near a normal host gene and provide an enhancer or silencer that changes expression of that host gene -- can turn on a proto-oncogene.

5. Cancer develops in stages

a. Most cancers have more than one mutation

b. Selection for increasing loss of growth control occurs as disease progresses -- cells that grow more aggressively (due to additional mutations) outgrow the others.

c. Normal cell --> benign tumor --> malignant (invasive) --> metastasis (spreads)

     C. Heritability -- can cancer be inherited?

1. You can inherit a predisposition to cancer, not the disease itself.

2. If you inherit versions of genes giving low repair or high mutability, tend to get cancer -- sooner or later, one of the random mutations that occurs messes up a proto-oncogene or tumor suppressor gene as above.

3. If you are heterozygous for an inactive blocking gene, you can get cancer if second copy gets messed up. (Example: retinoblastoma -- more details next time.)

4. Most cancer is sporadic, due to somatic mutation. Not inherited.

5. Most cancers not usually caused by virus. (There are exceptions: Cervical cancer is largely viral in origin.)

Next time: wrap up of cancer & on to hormones.