Lecture #3: Mechanism of Hormone Action--Secretion &Receptors

I. INTRODUCTORY REMARKS

1. Remember that the plasma membrane is like a mosaic (Fig. A):

(a) the membrane contains proteins, cholesterol [cholesterol], receptors, enzymes, ion gates, and signal transduction mechanisms.

(b) the phospholipid bilayer is fluid and dynamic (i.e., changing)

(c) the most common phospholipids include phosphatidyl-choline [phosphatidylcholine], phosphatidyl-ethanolamine [phosphatidylethanolamine], and phosphatidyl-inositol [inositol] (PIP₂) (also, see Fig. 3.B.A).

2. Ligands [ligand] (i.e., agonists & antagonists) are hormones (i.e., "first messengers") such as neurohormones, neurotransmitters, or other hormone-like substances (e.g., BK, PGs, PAF, GFs, etc.) that must first be secreted from their endocrine cells of origin, then be transported to the target cells, and finally react with membrane receptors on the target cells.

3. Keep in mind that phosphorylation [phosphorylation] (i.e., the addition of high-energy phosphates to a molecule such as an enzyme) is occurring extensively in all phases of cellular metabolism. The family of enzymes responsible for phosphorylation are called kinases [kinase], and many different protein kinases have been characterized.

II. SECRETION OF HORMONES BY ENDOCRINE AND NEURONAL CELLS

1. Secretion of peptide/protein hormones from an endocrine or neuroendocrine cell begins with synthesis of the hormone by ribosomes docked to the rough endoplasmic reticulum. (Fig. C)

a. the hormones are translocated to the lumen of the ER, where they can be glycosylated.

b. they travel through ER lumen toward the Golgi apparatus where they form secretory vesicles.

c. in ER and Golgi, they usually undergo proteolytic processing to a more bioactive form (Fig. 2.7)

d. eventually, these fuse into secretory vesicles, which travel along the cytoskeleton toward the inner edge of the plasma membrane. (Fig. D)

e. neurotransmitters are packaged in synaptic vesicles at axonal endings (Fig. E).

f. the vesicles are stored in proximity to the site along the membrane where they will be released.

2. Action potentials facilitate the secretion of hormones, neurohormones, and neurotransmitters.

(NOTE: Endocrine, neuroendocrine, and neuronal cells all they express action potentials.)

3. Action potentials (i.e., membrane depolarization) opens calcium ion channels in the plasma membrane and also cause the sudden release of calcium ions stored in the lumen of the ER.

4. The instantaneous increase in cytosolic calcium initiates exocytosis [exocytosis]) of vesicular contents.

5. Exocytosis involves specific interaction of SNARE proteins that mediate the fusion of phospholipids of the membrane of a secretory vesicle with the phospholipids of the plasma membrane of a cell.

6. The principal SNARE proteins involved in exocytosis are: (Fig. F)

a. synaptobrevin in synaptic terminals (along with cellubrevin in all cells) is a protein imbedded in the wall of a secretory vesicle that has a natural attraction for proteins in the plasma membrane.

b. syntaxin and SNAP-25, are the two principal proteins in the plasma membrane that have a natural tendency to form a stable complex with synaptobrevin (or cellubrevin), but this reaction is blocked by synaptotagmin.

c synaptotagmin is a second protein in the wall of the secretory vesicle that blocks the attraction of synaptobrevin to syntaxin and SNAP-25 in the plasma membrane.

7. Outlining the sequence of events involved in the process of exocytosis:

a. When the plasma membrane depolarizes in the vicinity of the small vesicles, the cytosolic Ca²⁺ concentration increases via voltage-gated Ca²⁺ channels (and Ca²⁺ release from the ER).

b. The Ca²⁺ induces a conformational change in the synaptotagmin, causing it to release the synaptobrevin and allowing this latter compound to complex with syntaxin and SNAP-25.

c. The fusion of the two membranes (i.e., the membrane of the small vesicle with the plasma membrane) leads to rapid opening of the vesicle to the extracellular fluids.

d. The time interval between influx of Ca²⁺ and exocytosis is about 200-300 m sec.

(NOTE: You might want to click HERE for a nice visual account of both exocytotic and endocytotic processes. As you look at this visual aid, think about the metabolic efficiency that is required to carry out the exocytotic reaction in only a fraction of one millisecond.)

III. INTERACTION BETWEEN HORMONES AND MEMBRANE RECEPTORS

1. Receptors provide the specificity for hormone-cell interaction.

2. At normal physiological levels, each hormone interacts with its own specific cellular receptor.

3. In most cells, a maximal biological response is achieved when only a small percentage (e.g., 1%) of receptors is occupied.

4. There are four major classes of membrane-bound receptors:

a. a “superfamily” associated with Guanine (GTP-binding) proteins. (GPCRs) (Fig 3.4.a)

b. receptors that are also enzymes (e.g., tyrosine kinase) (can span membrane once). (Fig. 3.4.b)

c. receptors coupled to ion channels, i.e., to ligand-gated ion channels.

d. receptors that bind cytokines [cytokine] and subsequently activate tyrosine kinase for signal transduction.

5. Most transmembrane receptors are proteins that span the plasma membrane 7X (Fig. 3.5, Fig. 14.9).

a. the membrane-spanning segments consist of 22-28 hydrophobic residues that have natural affinity for the phospholipid bilayer. (Fig. 9.15)

b. such receptors have a recognition site for ligand at the glycosylated N-terminus [glycosolation]. (Generally the longer the N-terminus, the larger the ligand for the receptor.) (Fig. 19.2)

c. most receptors for peptide hormones and neurotransmitters are linked to G-proteins at the third intracellular loop (in association with the C-terminus).. (Fig. 14.12)

d. Thus, this third loop and the C-terminus are relatively long in order to associate with G-proteins. In addition these segments of the receptor usually have phosphorylation sites.

6. Receptors are formed by the usual transcription and translation processes (i.e., like any protein).

7. Receptors are extraordinarily dynamic molecules (i.e., they change their shape, location, etc.)

8. Receptors can be phosphorylated by kinases, or they can auto-phorphorylate when a ligand couples with them. (But, this may reduce additional ligand binding.)

9. Receptors can be inclined to internalization and sequestration once they are phosphorylated.

10. Ligand/receptor coupling initiates membrane signal transduction processes by causing conformational changes in the receptor upon its phosphorylation, or auto-phosphorylation.

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