Discovery of G-proteins and the role of these proteins in signal transduction

Martin Rodbell & Alfred G. Gilman Wen-Chun Shaw

Dr. VanKleyScientific Discovery

What is G-protein??

(A)

(B)

(C) (D)

G-protein coupled receptor signaling

G-protein coupled receptor

GPCR and Disease

Cholera

Cholera is caused by a comma-shaped bacterium, Vibrio cholerae, which is ingested in contaminated water and food. The bacteria multiply enormously in the intestine, where epithelial cells allow fluid to leak into theintestine with intense diarrhoea as a result. Cholera is endemic in India and other parts of the third world.

• The bacterium discovered by Robert Koch in 1884, can be killed by antibiotics, but the disease is caused by a bacterial toxin, which irreversibly activates the G proteins of epithelial cells in the intestine. This results in an often life-threatening loss of water and salts. From Koch's discovery of the cholera bacterium in 1884 it took researchers about 100 years to expose the real cause of the disease - the effect of the bacterial toxin on G proteins

Inherited Night Blindness

• Scientists have found a G-protein defect in one type of inherited night blindness. Persons with this condition have a mutation in the gene that codes for the G-protein found in the eye's rod cells. Scientists believe that this defective G-protein is overactive. It stays turned on more than it normally would, and the person can't see well at low light levels.

McCune Albright Syndrome1. named for the two physicians who

described it over 50 years ago. 2. They reported a group of children, most of

them girls, with an unusual pattern of associated abnormalities.

3. In this disease, a mutation occurs sometime after conception, affecting only some of the body's cells.

4. Scientists have found that the mutation affects the same G-protein involved in cholera. This G-protein gets active continuously.

5. Skin cells, this causes darker pigment. 6. Bone cells, it causes weakness and

fractures.7. Hormone-producing cells the mutation causes the release of excess hormones

Martin Rodbell

The Nobel Prize in Physiology or Medicine 1994

• "for their discovery of G-proteins and the role of these proteins in signal transduction in cells"

• 1925 --born in Baltimore, Maryland, December 1st, son of Milton W. Rodbell, a grocery store owner

• 1943 -1943 --enters The Johns Hopkins University; studies biology and French literature

• 1944-1946 --education interrupted: drafted into the Navy; serves as a radio operator in the South Pacific, China, the Philippines, and Korea

• 1949 --receives B.S. in biology, The Johns Hopkins University • 1949-50 --post-graduate study in chemistry at Hopkins • 1950 --marries Barbara Charlotte Ledermann; four children;

seven grandchildren

• 1950 --moves to Seattle and enters Ph.D. program in biochemistry, University of Washington

• 1954 --completes Ph.D. thesis, Lecithin Metabolism in the Liver, under Donald H. Hanahan

• 1954-56 --postdoctoral position at U Illinois; research associate in biochemistry

• 1956-61 National Heart Institute, NIH as research chemist in Laboratory of Cellular Physiology and Metabolism

• 1960-61 --NIH-sponsored training at University of Brussels, Belgium, and Leiden University

• 1967-68 --Institute of Clinical Biochemistry, University of Geneva, professor and acting director

• 1971 --publishes core of work on G-proteins in a series of articles in the Journal of Biological Chemistry

• 1981-83 --Department of Clinical Biochemistry, University of Geneva, visiting professor

• 1985-89 --National Institute of Environmental Health Sciences, Chapel Hill, North Carolina, scientific director

• 1987 --inducted into the National Academy of Sciences; receives Richard Lounsbery Award.

• 1989-94 --NIEHS, Section on Signal Transduction, chief

• 1994 --retires; becomes NIH Scientist Emeritus; shares Nobel Prize with Alfred G. Gilman (announced Oct. 10, awarded Dec. 10)

• 1998 --dies at Chapel Hill, December 7

The concept of receptor Paul Enrlich (1854-1915).his work on immunity for which he was awarded the Nobel Prize for Medicine/Physiology in 1908 the development of selective chemotherapeutic agents, especially against syphilis and the foundation of haematology through his use of new dye staining techniques.

• In his voluminous thesis, Ehrlich proposed that the reactions between aniline dyes and cells was a chemical rather than a physical interaction, that there was a specificity between the dye and the cell or tissue it stains, and further that the chemical structure of the dye molecule defined its solubility and ability to attach (bind) to cells. As others have noted , here was the seed of a receptor theory.

Lock and Key

Isolation of single Fat Cells

• At that time, the only test medium available was crude chunks of fat tissue. No one can study the effect of hormones on individual cells.

• Because fat floats, Rodbell first put the minced tissue in a liquid and then treated the floating cells with a substance called collagenase. Then, fat cells flaoted to the surface ,and the stromal-vascular cells (capillary, endothelial, mast, macrophage, and epithelial cells)were sedimented.

Isolation of single Fat Cells

Second Messenger

• At that time, scientists knew that the adrenal gland produces epinephrine travels to body's cells and causes an increase in blood sugar. Let body have energy to react to stressful situations. But no one understood exactly how this hormone produced such an effect.

• In the late 1950s, Sutherland investigated the effect of epinephrine on liver tissue. He discovered that the hormone—the "first" messenger—stimulates formation of a "second messenger" within cells. It is this second substance, cyclic adenosine monophosphate (cAMP), that stimulates the breakdown of stored carbohydrate into sugar.  

• Rodbell realized that his isolated fat cells were the perfect medium for further investigation of the mechanism of hormone action.

Metabolism of Isolated Fat Cells

• Incubate isolated fat cells with Glucose-U-14C.

Comparison between tissue and Cells

Group(I) Unfed, ON Group(II) fed

Different hormone act on the fat cells

• Finally, Martin Rodbell create a system to analyze hormone action in individual fat cells.

• Even, different hormone can be used in this system.

• Many researchers began using Rodbell's method, making "The Metabolism of Isolated Fat Cells" one of the most widely cited in the field.

Demonstration of distinct Hormone Receptor

• The various hormones were tested at maximal and submaximal concentrations alone or combinedwith the other hormones. Synergy was seen with some combinations, but, most importantly, additivity of response was not obtained with maximal concentrations of the hormones. Although not completely proof,they argued that it is likely that the fat cell cyclase system consists of multiple receptors interacting with a common catalytic unit.

• Because of the experimental complexity of studying the multi-receptor adenylate cyclase system in rat adipocytes. Rodbell turned his attention to the glucagon-sensitive adenylate cyclase system in liver.

Informational processing: the concept of transduction

Glucagon-sensitive adenylate cyclase system

in liver.• Chromatography of 125I-Glucagon• Measurements of Adenyl Cyclase Activity-

measured by the conversion of 32P-ATP to cyclic 5’-AMP

• This system can investigate both the nature of the glucagon receptor and the relationship between hormone binding and hormonal activation of adenylate cyclase.

The actions of GTP and Glucagon on Liver Cyclase

• Rodbell discovered that ATP could reverse the binding action of glucagon to the cell receptor and thus dissociate the glucagon from the cell altogether.

• GTP could reverse the binding process almost one thousand times faster than ATP

• This GTP, he found, stimulated the activity in the guanine nucleotide protein (later called the G-protein) in the cell

GTP Hydrolysis

• A few months later, they found that Gpp(NH)p caused the enzyme’s activity to “take off’ to an extent not even seen.

General Characteristics of Guanine Nucleotide Action

-subunit uniquely capable of binding and degrading

GTP and a tightly knit complex of and

subunits.

α β

γ

The future of GPCR

Valuable G-protein coupled Recptor

GPCRs are good drug targets 50% of subscription drugs interact

with GPCR• Hypertension• Stomach ulcers• Migraine• Allergies

GPCRs in disease states Disease states associated with GPCR mutations•Rhodopsin receptor retinitis pigmentosa

•Vasopressin V2 nephrogenic diabetes•Glucagon diabetes, hypertension

Reference

• www.nobel.se• http://history.nih.gov/• http://profiles.nlm.nih.gov/• Metabolism of Isolated Fat Cells, Martin Rodbell, J. of

Biol. Chem., , Vol.239, No. 2, February 1964• The Glucagon-sensitive Adenyl Cyclase System in Plasma

Membranes of Rat Liver, J. OF Biol Chem., Vo1.246. No.6, pp. 1857-1869,1971, Lutz Birnbaumer, Stephen L. Pohl, and Martin Rodbell

• The Fat Cell Adenylate Cyclase System, J. OF Biol Chem.,Vol 254 ,No.18, pp8927-8931, 1979, Dermot M. F. Cooper, Werner Schlegel, Michael C. Lin, end Martin Rodbell

• The role of hormone receptors and GTP-regulatory proteins in membrane transduction, Nature, Vol. 284, No. 575 1, pp. 17-22. March 6 1980, Martin Rodbell

• 5'-Guanylylimidodiphosphate, A Potent Activator Adenylate Cyclase Systems in Eukaryotic Cells, Proc. Nat. Acad. Sci. USA Vol. 71, No. 8, pp. 3087-3090, August 1974, Constantine Londos, Yoram Salomon, And Martin Rodbell