C2006/F2402 '09 Outline for Lecture 22 --  (c) 2009 D. Mowshowitz  -- Lecture updated 04/24/09

Note: Some minor changes in format & in problem numbers were made after the morning lecture. The changes are highlighted in blue.

Handouts  From last time: 21C -- Thyroid gland & thyroid hormone

New this time:  22A -- Lactation; 22B -- Stress Response; 22C = Basic Processes in Kidney Tubule

I. Hormones, cont. -- More on HT/AP Axis

1. General case:  See Sadava fig. 41.8 (42.8)

a. The cascade: 

b. Negative FB: Hormone from target gland (thyroxine, sex steroids, etc.) has negative feedback effect on  AP (& also in some cases on HT).

2. Specific case: thyroxine production (See handout 21C)

a. The cascade:

b. Regulation

(1). Negative Feedback: TH inhibits production of both TSH and TRH. (Where are the receptors? On cell surface or intracellular??) Primary effect is at AP -- reduces response to TRH.

(2). Two different types of goiter (enlarged thyroid)

(4). Note that levels of TH, TSH & TRH are regulated, unlike case with insulin.

1. What does thyroxine do? Raises BMR and is needed  for normal alertness and reflexes. Needed during childhood for brain development.  See In Raising the World’s I.Q., the Secret’s in the Salt This article is part of a series from the NY Times (2006). Articles in this series examine diseases that hover on the brink of eradication, and the daunting obstacles that doctors and scientists face to finish the job.

Here are two more recent articles on Thyroid Function from the Times:
    Prenatal Testing of Thyroid Debated
    Raising the World's IQ (an op ed)

2. Biochemistry: Structure of TH -- How modification and rearrangement of tyrosines in thyroglobulin (TG) leads to TH. (See handout.)

3. Cell Biology: How is thyroxine made & stored?  How (& where) TG is made & TH released from it. 

• TH made in follicle of thyroid gland

• protein (TG) made on RER → Golgi → vesicles

• exocytosis of vesicles releases TG into lumen

• I- taken up into gland; I added to tyrosines of TG in lumen; one modified tyrosine added to OH of another. 

• Modified TG stored in lumen of gland = reservoir of TH

• TG taken up by cell from gland by RME. Result is degradation of TG in lysosomes → releases T4 or T3 (= TH)

• TH  diffuses out of cell across membrane. Acts like a steroid. (For structures see handout or texts.)

• TSH stimulates virtually all of these steps.

4. How does thyroxine travel through the blood? All lipid soluble hormones are attached to plasma proteins, either to general proteins or specific binding proteins for that hormone. T4 and T3 are transported by thyroxine-binding globulin, which is specific for thyroxine. Note: thyroglobulin is not the same as thyroxine-binding globulin. (Globulin just means globular, soluble protein.)

Try problem 7-5 & 7-9. (If you have time, there are additional problems on this topic -- most of problem set 7. )

Try problems 7-15 & 7-19.

1. Phase one -- Nerve (Sympathetic) activity stimulates target organs (that are not glands) → Direct response of heart, liver, lungs, etc.

2. Phase two -- Nerve (Sympathetic activity) → activation of glands

a. Stimulate pancreas → glucagon release stimulated; insulin release inhibited → secretion of glucagon → additional stimulation of some of same target organs

b. Stimulate adrenal medulla → release of epinephrine → stimulation of same targets as sympathetic nerve activity & some additional targets -- hormones can reach where nerves can't go.

3. Phase three -- stimulate HT/AP axis to produce cortisol

 HT in brain → releases CRH → AP → releases ACTH → adrenal cortex → produces cortisol → target organs → stimulation of breakdown of fats & protein for energy (sparing glucose for brain); inhibition of immune system.

Note that each additional phase is slower but involves additional degrees of amplification due to second messengers, transcription, etc.

III. Signaling with RTK's. How do RTK's Work? 

    A. Importance of Catalytic Receptors

1. What are they? Catalytic Receptors are surface receptors whose intracellular domain is an enzymatic domain (or binds to one)

2. How do they work? Ligand binding activates the enzymatic domain. The active enzyme modifies a cellular protein, which binds to or modifies other proteins, etc.

3. Types: The enzymatic domain is usually a kinase, often a tyrosine kinase. We will stick to receptor tyrosine kinases & TK linked receptors.

4. What signaling molecules use TK receptors?

• Many growth factors (such as EGF) and other paracrines, autocrines and juxtacrines act through TK receptors.
• Insulin, PL, and GH, but not most other endocrines, act through TK receptors. (See Sadava 15.6)

5. Results: 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.)

    B. Important Properties of Receptor TKs -- See handout 22B & chart below.

1. Receptor is usually a single pass protein 

2. Ligand binding usually leads to dimerization of receptors (Sadava fig. 15.6 or Becker 14-17)

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. (Sadava fig. 15.10 (15.9), or Becker 14-18)

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

• Example? PLC. Ligand binding to TK-linked receptor can activate a type of PLC → IP3 & DAG → etc.
• 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.  

b. Indirect Effect: 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  phosphorylated TK (to the phosphorylated tyrosines)

(2). Additional proteins bind to the adapters 

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

(4). Most famous (infamous?) example is the small G protein ras. Why does anyone care about ras?

(a). Overactive form of ras (stuck in the activated 'on' form) can cause cancer. Implies normal ras involved in control of cell cycle. (More on ras in optional lect. 25.)

(b). Large % (about 30%) of tumors have overactive ras.

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

d. Recruitment. Note that the initial target protein(s) to be activated come to or are "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.

    C. A human example of TK receptor signaling:  FGF (Fibroblast growth factor) and FGF Receptor.  Significance of dimerization. See Becker figs.14-20 & 14-21 (14-19 & 14-20).

1. FGFR is a TK receptor  

2. FGF & FGFR needed for proper development as described in Becker chap.14. Failure of signal transmission (or premature transmission) causes developmental abnormalities. See Becker fig. 14-20 (10-20).

3. How can achondroplasia (a type of dwarfism), due to a defective FGF receptor, be dominant? See Becker fig. 14-20 (14-19), and box on handout 22B.

a. The general principle: TK receptor monomers must dimerize in order to act. If 1/2 the receptors (1/2 the monomers) are abnormal, most of the dimers that form are abnormal.

b. An important consequence: "lack of function" mutations in TK receptors are often dominant. (For an example see Becker figs. 14-20 & 14-21 (14-19 & 14-20). Dimers form, but they are inactive.

c. Definition: An abnormal allele like the one that produces the FGF receptor without a cytoplasmic domain 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).

d. Significance: What does the existence of a dominant negative mutation imply? It indicates that the gene involved probably codes for a protein that must polymerize in order to act.

e. This case: In a heterozygote for achondroplasia, 1/2 the FGF receptor (monomers) are defective, therefore dimers that form are defective.

(1). In the example shown in Becker & on handout,  the cytoplasmic domain of the defective receptors is missing. Dimers form, but most dimers are never activated -- the two monomers can not phosphorylate each other. (Fig. 14-19) Therefore the signaling is badly disrupted. (In the example shown in Fig. 14-20, the FGF is needed to turn on formation of certain embryonic tissues, so the mutant fails to form these tissues. This type of mutation is called a 'dominant negative' as explained below.)

(2). (FYI)  In most cases of human achondroplasia, the molecular explanation is different. In these cases, the mutation is in the transmembrane domain of FGF3 Receptor and dimers form, but act abnormally. (In these cases, the FGF signal is needed to turn on bone differentiation and turn off cell growth. Mutant dimers signal prematurely, so differentiation starts -- and cell growth stops -- before bones are long enough. This type of mutation is called a 'gain of function' mutation, because it works when it shouldn't.)

4. Dominant Negatives -- Some Additional Background

a. 'Dominant negative' means that the heterozygote is negative for function, not that it doesn't produce any protein. There is actually a mixture of normal and abnormal protein present. What's unusual is that the abnormal, inactive, protein 'gets in the way' and interferes with the working of the normal, active, protein. So overall, there is a lack of function in the heterozygote.

b. Negative mutations (those that produce inactive protein) are usually NOT dominant.  Most negative or "lack of function" alleles (or mutations) are recessive. If there is a mixture of normal and abnormal protein in the heterozygote, the normal, active, protein usually works (in spite of the presence of abnormal, inactive, protein). So usually, overall, there is NO lack of function in the heterozygote.

    D. How do GPLR's and RTK's Compare? -- See table below for reference for comparison of basic features of  TK or TK linked receptors and G protein linked receptors.  For TK's, see Sadava figs. 15.6 & 15. 10 (15.9) or Becker fig. 14-17 for structure and Becker 14-18 for signaling pathway.

Review problems 6-1 & 6-3

    E. Signaling Pathways all interrelate

1. Different 2nd messengers can influence the same enzyme/pathway. See problem 6-20.

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

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. 14-18).

IV. Intro to Kidney Structure & Function (Handout 22C ). See also Sadava Sect. 51.4. & 51.5

e. Cells in different parts of tubule have different transporters/channels on luminal surface.

f. Cells in different parts of tubule (except maybe descending loop) all have the Na⁺/K⁺ pump on their basolateral surface. Other transporters may vary.

g. Depending primarily on which transport proteins are on luminal surface, cells secrete materials into lumen or reabsorb material from lumen.

h. Cells lining tubule do actual secretion/reabsorption but peritubular capillaries remove reabsorbed material or provide material to be secreted. Therefore (as shown on handout 22C, top left):

(1). Result of tubular reabsorption = net transfer from filtrate to capillary.

(2). Result of tubular secretion = net transfer from capillary to filtrate.

i. For an example of reabsorption -- see 22C, upper right. (Fig. 14-18).

Try problem 12-3.

3. Role of Hormones

a. Affect retention of water and/or Na⁺ near end of kidney tubule.

b. Cause water and/or Na⁺ to be removed (reabsorbed from filtrate)

• aldosterone affects Na⁺  reabsorption (& K⁺secretion)

• ADH affects water reabsorption

c. Role/Mech. of action of ADH

(1). ADH (using cAMP) stimulates insertion of water channels/pores into membranes of cells lining part of tubule

(2). Water flows out ADH-stimulated channels (if in membrane) because of salt gradient in interstitial fluid surrounding tubule

(3). Diabetes insipidus -- result of no ADH or no response to ADH

d. Role of aldosterone in Na+ reabsorption

(1). Promotes reabsorption of Na⁺

(2). Amount of Na⁺ reabsorbed due to aldosterone is small % of total, but adds up.

e. Questions to think about: Where are the receptors for ADH? Aldosterone? Which hormone elicits a faster response?

Next Time: We'll look at overall structure of kidney, and follow some liquid through to see where basic processes occur;  then: Intro to the Immune System.