C2006/F2402 '11 OUTLINE OF LECTURE #14

(c) 2011 Dr. Deborah Mowshowitz, Columbia University, New York, NY. Last update 03/07/2011 06:38 PM.  A few minor formatting changes made 3/9 am.
Handouts:  14A --  Overview of Signaling  -- the Biological Big Bang Theory
                  14B --   Structure of G proteins and GPCR's; cAMP pathway
                  14C -- Comparison of Organelles -- Lysosomes, Peroxisomes & Mitochondria

Some Interesting/Useful Links
Discovery of G proteins. The 1994 Nobel Prize was awarded to Gilman and Rodbell for discovering G proteins and the processes of signal transduction.  The "Press Release" describes their scientific findings, and there are neat illustrations and biographical notes.  

A hypertext of endocrinology with some animations is at
http://arbl.cvmbs.colostate.edu/hbooks/pathphys/endocrine/index.html

Several animations of Signaling are at the McGraw Hill Web site for the Raven text book.
See the Web-Links page for more links to animations & other online resources

I. Peroxisomes -- summary of structure, function and synthesis.  For more details see Becker  pp. 356-360 (354-358). How do proteins reach organelles that are not part of the endomembrane system?

    A. Structure -- comparison of peroxisomes and lysosomes

1. Summary Table -- See handout 14C. Features to consider:

1	Membrane(s) around organelle -- How many?
2	Contain DNA?
3	Grow & Split?
4	Where are organelle proteins made? Where are the ribosomes?
5	Protein import is co-translational or post-translational?
6	Localization Signal #1 =?
7	Additional LS?
8	Function of organelle?

2. How are peroxisomes distinguished from lysosomes, since both organelles are so similar in structure?  

a. Biochemical Method: Peroxisomes separated from lysosomes biochemically ("grind and find" method) after growing animals on diet with triton (detergent). Details:

• Lysosomes accumulate detergent.

• Detergent = mimic of  phospholipid = amphipathic molecule with hydrophobic and hydrophilic ends. 

• Density of organelles is proportional to protein/lipid ratio. Growth on detergent alters the ratio in lysosomes.

• Lysosomes with triton (equivalent to extra lipid) are unusually light (low density, because of high lipid/detergent content).

• Density of peroxisomes is unchanged by growth on detergent.
   

b. In situ Method: Peroxisomes identified in situ as different from lysosomes by marker enzymes

•  Marker enzymes = enzymes characteristic of and unique to a particular organelle.

• Typical marker enzymes for peroxixomes are urate oxidase or catalase

• Typical marker enzyme for lysosomes is acid phosphatase

    B. Major Function = detoxification (in animal cells). See Becker for other roles, esp. in other organisms.

1. Role of Oxidases: Oxidases catalyze: 

RH₂ + O₂ → R + peroxide (H₂O₂)

• Oxidation detoxifies RH₂ by decreasing toxicity, and/or by increasing solubility

• R is generally more soluble and less hydrophobic than RH₂.

• Soluble material is more easily transported through blood** and excreted by the kidneys; hydrophobic material is more likely to accumulate in the body (& reach toxic levels).

• Note that these reactions are real oxidations (involve actual addition of oxygen) not dehydrogenations (= removal of H's and electrons) as in most of energy metabolism.

• These reactions generate peroxide, which is very reactive.

** Cells that carry out oxidations generally have transporters to allow soluble material to exit cell and enter blood.

2. Role of Catalase: Catalase catalyzes:

  H₂O₂ + R'H₂  → R' + 2 H₂O

R'H2 can be a second molecule of peroxide.  In that case, R' is oxygen and overall reaction is: 

   2 H₂O₂ → O₂ + 2 H₂O

Net Result:

• Catalase gets rid of peroxide, and

• Catalase generates oxygen for another go round of oxidation (if R'H₂ is peroxide) or detoxifies R'H₂ (by oxidizing it)

    C. How do Proteins (& phosopholipids) get into Peroxisomes? Here is a summary of the details:

1. Matrix Proteins

• Peroxisomes are not considered part of the endomembrane system --  all the proteins of the matrix are made on cytoplasmic free ribosomes.

• All proteins of the peroxisomal matrix enter the organelles post translationally. Matrix proteins go directly to peroxisomes from free ribosomes. 

•  Localization signals for best known peroxisomal matrix enzymes are on COOH end of protein. (Some perox. proteins have the signal elsewhere; not all signals are the same. See problem 3-19, esp. part C.) 

• Mechanism of import is not well understood -- some matrix proteins enter without unfolding. Entry requires ATP.

2. Membrane lipids

• Some phospholipids of peroxisomal membrane are made on the ER. Carried to peroxisome by transport/exchange proteins and/or vesicles.

• Some lipids are made in the organelle.

3. Membrane Proteins

• The proteins of the membrane and the proteins of the matrix enter the organelle by separate pathways.

• Some membrane proteins may be made on the ER, but this is not settled.

4. How do new peroxisomes form?

• Peroxisomes have no DNA, and have been seen to multiply by growth and division.

• Under certain circumstances, vesicles from the ER are able  to give rise to new (empty) peroxisomes de novo. If this happens, all the matrix proteins must be imported.

• In summary: All perox. matrix proteins are made on free ribosomes, and most experiments favor the growth and division model as the main source of new peroxisomes.

To review how proteins enter peroxisomes, try problem 3-7.


II. Mitochondria & Chloroplasts  Where do their proteins come from?

    A. Some Proteins are made inside the organelles

• Some proteins (not many) are made inside mitochondria using organelle DNA and organelle transcription and translation machinery.  

• Mito make their own rRNAs & some or all tRNAs, but the rest of the transcription and translation machinery comes from outside.

• Chloroplasts (in plant cells, in addition to mitochondria) have more DNA than mitochondria, and make more components inside than mito., but most chloroplast proteins are also imported from outside.

    B. Most organelle proteins must be imported.

1. Most organelle proteins are made on free ribosomes and then imported (post-translationally) into the organelles. 

2. Organelle Membranes contain translocases. Proteins are imported by passing through pores or transport complexes (translocases) in the organelle membranes. See Becker 22-18.

3. How to reach the matrix? Proteins can pass through both mito. membranes at once. Proteins enter matrix by crossing membranes at contact point = point where membranes are very close together and translocases of the inner and outer membrane are aligned. See Becker fig. 22-19.

4. Localization signals.

• A short sequence called a transit peptide (TP) on the amino end is the usual localization signal that targets a protein to attach to a mito. translocase and enter the matrix.

• The TP is removed once the protein enters the matrix of the mitochondrion. (Additional signals are required to direct the protein to a membrane or the intermembrane space. See 7 below.)

• Similar sequences are used to direct chloroplast proteins to translocases on the organelle and to the correct organelle subcompartment.

5. Entry requires energy in form of ATP and/or electrochemical (proton) gradient. Note that transfer is post-translational so energy of protein synthesis cannot be used to drive entry of protein into organelle.

6. Chaperones (chaperonins) are needed.  Proteins are translocated in an unfolded state. Every time a protein remains/becomes unfolded to cross a membrane (or refolds on the other side) a chaperone is needed. Two or more types of chaperones involved here -- one in cytoplasm and one or two in matrix of organelle. See Becker fig. 22-20.

a. Chaperones in cytoplasm keep protein unfolded or loosely folded until it reaches mitochondrion.

b. Chaperones inside organelle may help "pull protein in" and help protein fold properly once it enters. 

c. Release of chaperones requires hydrolysis of ATP.

7. How do proteins reach parts of the organelle other than the matrix?

a. Additional localization signals and/or stop/transfer sequences are required -- in addition to a transit peptide. Different proteins probably use different localization signals and/or pathways.

b. One approach -- from the outside: Proteins destined for the membranes or the intermembrane space may enter the outer membrane, cross only part way in, and never reach the matrix -- the proteins could lodge in the appropriate membrane (if they have a 'stop' transfer or hydrophobic anchor sequence) or stay in the intermembrane space.

c. An alternative approach -- from the matrix: Proteins destined for sites other than the matrix may enter the matrix first, and then use a hydrophobic 'start' sequence to cross back out to the membranes or intermembrane space from the matrix. (See problem 3-6.)

8. Summary Charts. See handout 14C for: 

    a. Top --Comparison of Mitochondria vs Lysosomes & Peroxisomes

            b. Bottom -- Comparison of Features of Peroxisomes to those of Mitochondria vs Lysosomes

Want to be sure you've got this down? Make an empty chart like 14C (top and/or bottom) with only the headings and categories provided, and then fill it in without consulting your notes or texts. Look up anything you can't remember, and try it again in a few days.

III. Introduction to Signaling  

    A. Big Issues

1. What does signaling involve? How are messages sent from one cell to another? How are they received -- how do signals produce a response in the target cell?

2. Why bother with signaling? It's needed so events in a multicellular organism can be coordinated. It's not enough to regulate what one cell does!

    B. Usual Method -- one cell secretes signal molecules that bind to a receptor on (or inside) a target cell → amplification →  big effect in target cell. 

    C. Major types of Signal molecules -- classified by type of cell that makes them and/or target location. Where do the signals come from, and where do they go? See Handout 7B for pictures & Lecture 7 for details.

Good way to study this: Make a table summarizing info on handout 7B. Include name of type of signaling, source of signal, type or location of target cell, any other important features, and an example of each.

    D. How do secreted signal molecules work at molecular level? Overview. See handout 14A, bottom.

1. Signal Molecules

a. Signals are evolutionarily conserved. Same signal molecules used by different organisms or cell types for different purposes.

b. Two main kinds of signal molecules

(1).  water soluble

(2).  lipid soluble.

Question: Which types of signals are usually released by exocytosis?

2. Receptors.

a. First step in signaling is binding (noncovalent) of signal to receptor, causing conformational change in receptor.

b. Locations of Receptors -- intracellular and on cell surface. See Sadava fig. 7.5 (15.4).

(1). Intracellular -- for lipid soluble signals. All similar, all TF's -- details below

(2). On Cell Surface -- for water soluble signals. See Becker fig. 14-2.

(a) Structure: All cell surface receptors are transmembrane proteins with an extracellular binding domain for signal.

(b). Terminology: Cell surface receptors are sometimes called "extracellular receptors" but only ligand-binding domain is extracellular, not the entire protein.

(c). Ligand binds to extracellular domain on the outside; effect ('big bang') is inside the cell.

Signal Type	Example	Receptor Type	Effect
Lipid Soluble	Thyroxine, steroids	Intracellular**	Gene activity
Water Soluble	Peptide hormones, GF's	Cell Surface	Protein activity (usually)

**Note: Some lipid soluble signals have cell surface receptors in addition to their intracellular receptors. One such case is in the problem book. Cell surface receptors for lipid soluble signals have been discovered relatively recently, and will largely be ignored in this course.

    3. Amplification or the Biological Big Bang.  All signals → amplification = big effect from a small concentration of signal. Example: 1 molecule of epinephrine can cause release of 10⁸ molecules of glucose from a liver cell ! (See Becker 14-3 for the calculation.)

    4. How is amplification achieved?  How is the 'Big Bang' Accomplished? Three ways ( See handout 14A):

a. By opening (ligand-gated) channels

(1). General Idea:

ligand binds

→

open a few ligand gated channels

→

a little ion flow

→

hit threshold voltage

→

open many (voltage-gated) channels

→

big change in ion concentrations

→

big effect

                (2). Specific example: Acetyl choline (AcCh) effects on muscle. Signaling by AcCh is important in both muscle & nerve responses. AcCh receptor is a Na⁺ channel in plasma membrane opened by AcCh.

AcCh binds

→

open a few ligand gated Na+ channels

→

a little Na+ flows in

→

cell becomes less - inside; hits threshold voltage

→

open many (voltage-gated) Na+ channels

→

big change in Na+ concentrations

→

Muscle contracts

Note: Specific examples are here primarily for reference. More details of each specific example will be discussed below or in later lectures.

b. By cascades of modification  →  lots of (pre-existing) protein is modified→  big effect.

    (1). General idea:

ligand (1st messenger) binds

→

activate receptor in membrane

→

activate protein inside cell (usually a chain of activations = cascade*)

→

activate a lot of target protein (enzyme,  or TF, etc.)

→

lots of product

            (2). Specific Examples: TSH & epinephrine (2 hormones). TSH stimulates release of thyroid hormone from thyroid gland. Epinephrine stimulates glycogen breakdown. Many water soluble hormones work in this way.  Details of receptors, cascades etc. later.

* The first example for this type of modification cascade to be discovered was the breakdown of glycogen, stimulated by the hormone epinephrine (adrenaline). For details on the extent of amplification, see Becker fig. 14-3 or Sadava fig. 7.20 (15.18).

c. By affecting transcription/translation →  lots of new protein made →  big effect

ligand binds

→

activate a TF

→

transcribe a gene

→

make lots of mRNA molecules/gene

→

mRNA translated

→

many new protein molecules/mRNA

Example: Thyroid hormone (also called thyrotropin or TH) & steroid hormones. Most lipid soluble hormones act in this way. Receptor is itself a TF.

Question: How does the speed of signaling compare for the three types of amplification?

Try problems 6-12 & 6-13.

    E. Summary of important issues of signaling to consider (so far) -- See handout 14A.

1. Receptors -- cell surface vs internal (Sadava, fig. 7.5 (15.4))

2. Signals -- lipid soluble vs water soluble

3. Amplification -- 3 basic methods

IV. How do Intracellular Receptors Work? See Sadava fig. 7.9 (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

• 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/basal promotor -- can be considered part of (core) promotor, or as separate proximal upstream sites.

    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 TF receptors bind their lipid soluble ligands  -- how receptors are activated & affect transcription

1. Binding -- Receptor binds its ligand

2. Disassociation -- Receptors disassociate from inhibitory proteins.    

3. Dimerization -- Receptors dimerize -- form pairs.

4. Location -- If receptor is in cytoplasm, moves to nucleus.

5. DNA binding -- Activated Receptor (dimerized & bound to ligand) binds to HRE on DNA.

6. 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.

*Details vary somewhat with different hormones (& corresponding receptors)

    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 (multiple) target genes. Binding → increased transcription of some genes (genes activated); decreased transcription of others (genes 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). 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).

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 such as 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.

V. Types of Cell Surface (Transmembrane) Receptors (See bottom of 14A)

    A. Channels. Some receptors are themselves (parts of) channels. (See AcCh receptor above and Sadava fig. 7.6 (15.5) Other receptors are not channels, but work by opening or closing separate channels. Channels will be discussed at a later date by Dr. Firestein.

    B. Types of Cell Surface receptors that are not channels

1. Type 1: Linked to G proteins.

a. Terminology: Called G protein linked receptors, or G Protein Coupled Receptors (GPCRs). For a generalized case, see Sadava fig. 7.8 (15.7).

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

c. What are they receptors for? Many hormones such as use TSH  & epinephrine use GPCRs.

d. How do they work? Activate a G protein, which acts as a switch to trigger amplification (details below). G proteins usually: 

(1). Activate enzymes that generate second messengers (see Sadava fig. 7.8 (15.7)), or

(2). Open/close ion channels.

2. Type 2:  Not linked to G proteins. To be discussed in more detail later when we get to cell cycle and cancer. For reference:

a. Many are protein kinases. In addition to extracellular, ligand binding domain, have an intracellular kinase domain, or interact with an intracellular kinase (when activated).

b. Most well known type: Receptor Tyrosine Kinases (RTKs) -- also called  TK linked receptors.

c. Structure: Usually are single pass proteins that aggregate into dimers when activated.

d. What are they receptors for? Many Growth Factors use TK linked receptors or related receptors. (See Becker table 14-3 if you are curious).

e. How do they work? These usually generate cascades of modifications, but do not always use 2nd messengers.  If you want to see an example, see Sadava figs. 7.6 & 7.12 (15.6 & 15.10). We won't get to details of how these work for a while.

 VI. How do GPCRs & G Proteins Act in Signaling?

    A. Typical Pathway (see also handout 14B, middle panel)

 Note that the ligand (1st messenger) binds to the extracellular domain of its receptor. The remaining events are inside the cell.

    B. Second messengers -- See handout 14B or Sadava fig. 7.8 (15.7)

        1. What are they? Small molecules or ions that move through the cell and bind to their target proteins.

2.  The usual second messengers -- see handout 14B for structure of cAMP and mode of action

3. What do 2nd messengers do? Bind to and thereby activate (or inactivate) target proteins.

4. How they are made: Active G protein (subunit) → binds to & activates enzyme in (or associated with) membrane → generates second messenger in cytoplasm. See Becker fig. 14-7 or Sadava  figs. 7.8 & 7.14 (15.7  & 15.12) for cAMP pathway. We will get to IP3 pathway later. If you are curious, see Becker fig. 14-10 or Sadava fig. 7.15 (15.13).

5. A Specific Example: Thyroid stimulating hormone (TSH) -- promotes release of thyroid hormone (TH). 

        a. Generation of 2nd messenger (cAMP)

TSH (1st messenger) binds

→

activate GPCR in membrane

→

activate G protein in the membrane

→

activate enzyme in membrane (adenyl cyclase)

→

generate small molecule (2nd messenger) inside cell = cAMP

        b. Action of 2nd messenger

cAMP

→

activate protein kinase (PKA) in cytoplasm

→

phosphorylate target enzymes

→

stimulate multiple steps in synthesis and release of TH

        6. Why bother with all these steps?

            a. Amplification. Many steps involve amplifications. For example, one molecule of active Adenyl cyclase can generate many molecules of cAMP and one molecule of PKA can phosphorylate many molecules of its target enzymes. For an example of the possibilities of a cascade of amplification, see Becker fig. 14-3 or Sadava fig. 7.20 (15.18). (They don't agree on the exact numbers.)

            b. Examples. The usual example for this type of modification cascade is the breakdown of glycogen, stimulated by the hormone epinephrine (adrenaline), which is the example in Becker fig. 14-3.   If you are interested in more details of the pathway, see Sadava fig. 7.20 (15.18) or Becker fig. 14-25 (14-24). This example was the first to be discovered, but is more complex than the TSH case.

VII. How do G proteins Work?

    A. What are the important properties of G proteins? (See Becker fig. 14-5 & Handout 14B)

1. Have active and inactive forms

        a.  Active form is bound to GTP

        b.  Inactive form is bound to GDP.

2. G-proteins act as switches in many processes (not just signaling)

        a. Activation: G protein is activated by dumping GDP and picking up GTP in response to some signal.

        b. Inactivation: G protein inactivates itself by catalyzing hydrolysis of GTP to GDP.

        c. Why is it a switch? The G protein does not stay active for long. "Turns itself off."

    B. Activation & Inactivation of G Proteins

    C. Types of G proteins

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

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

b. Separation occurs on activation: On activation, alpha subunit (with the GTP) separates from other 2 subunits.

c. Either part  may be the effector that actually acts on target -- alpha, or beta + gamma, can act as activator or inhibitor of target protein.

d. Hydrolysis causes reassociation. Hydrolysis of GTP to GDP causes alpha to reassociate with other subunits → inactive heterotrimer

    D. For reference: Comparison of Protein Kinases, Receptor Protein Kinases, & Trimeric G proteins

Protein	Catalyzes	What's added to Target Protein?	Who gets the P or GTP?	How Inactivated?
Protein Kinase	Protein + ATP → ADP  + protein-P	Phosphate	Usually a dif. protein	Separate Phosphatase removes P
Receptor Protein Kinase**	Protein + ATP → ADP  + protein-P	Phosphate	Usually  separate subunit of self	Separate Phosphatase removes P
Trimeric G Protein	Exchange & Hydrolysis as described above.	GTP	Itself	Hydrolyzes GTP to GDP (by itself)

**Receptor protein kinases have an extracellular ligand binding domain and an intracellular kinase (or kinase binding) domain. Ordinary kinases usually add phosphates to other proteins. Receptor kinases usually add phosphates to themselves. (For an example, see Sadava fig. 7.7 (15.6)

Try problems 6-1 & 6-2.

VIII. An example of a second messenger -- cAMP  & its target (PKA)  See handout 14B or Becker fig. 14-7.

    A.   How is cAMP level regulated? What does it do?

    B. How do hormones work through cAMP?

1. TSH -- see above. PKA phosphorylates (and activates) enzymes needed to make thyroid hormone.

2. Glycogen metabolism:  This case is very complex and will be discussed later. If you want to read ahead, see Sadava fig. 7.20 (15.18) or Becker figs. 14-25 (14-24) & 6-17 (6-18 ).

Next Time (after break): Wrap up of chemical signaling; how signaling is used to maintain homeostasis.