Now researchers at the University of Wisconsin Medical School and the National Institutes of Health have determined the three-dimensional structure of the "catalytic core," or main power center, of adenylyl
cyclase, a critical enzyme in the initial signaling system.
The new structural information is an essential first step in the future development of drugs to treat abnormalities related to the enzyme, possibly including Alzheimer's Disease and some neuropsychiatric
Directed by Dr. Arnold Ruoho, UW Medical School professor and chair of pharmacology, and Dr. James Hurley, X-ray crystallographer at the National Institutes of Health, the research is reported in the
March 20 issue of Nature.
"The research of Drs. Ruoho and Hurley dramatically increases our understanding of how this important enzyme works, and provides clues to synthesis of drugs that could target it specifically," said
Dr. Allen Spiegel, scientific director of the NIH's National Institute of Diabetes and Digestive and Kidney Diseases, which together with the National Institute of General Medical Sciences, funded the research.
Adenylyl cyclase is a key player in the signaling system that receives messages from outside cells and sends them repackaged to cell centers involved in any number of activities.
Activation of the system begins when a "first messenger" in the form of a hormone or neurotransmitter outside the cell couples with a specific receptor in the cell membrane. The alerted receptor triggers changes in an adjacent molecule called the G-protein, in turn stimulating production of adenylyl cyclase (AC), which also sits in the cell membrane.
Once AC is turned on, it generates a "second messenger," called cyclic AMP, which tells specific cellular proteins to activate another part of the system.
"Activities as varied as heart muscle contraction, liver metabolism and hormone secretion all rely on this system to relay and transform signals outside cells to downstream 'production centers' inside
cells," said Ruoho.
Besides changing an extracellular signal into an intracellular one, the system also greatly amplifies the initial signal, said the UW molecular pharmacologist, who has studied all parts of the system
for more than 20 years.
Two Nobel Prizes have been awarded for studies related to the system, found in almost all cells in humans and animals, and in some microorganisms. Dr. Earl W. Sutherland's discovery of cyclic AMP
won him the coveted recognition, and Drs. Alfred G. Gilman and Martin Rodbell share the prize for their work with G-proteins.
Ruoho was intrigued with recent observations by Gilman and his former associate, Dr. Wei-Jen Tang, who found that two small strands of adenylyl cyclase, called C1 and C2, form the AC catalytic core
and are responsible for generating cyclic AMP. The strands appear to function inside the cell away from the bulk of the protein sitting in the membrane.
Ruoho contacted NIH X-ray crystallographer Hurley in order to learn more about the three-dimensional molecular structure of C2, which is very similar to C1 and which the UW researchers showed can independently produce cyclic AMP in some circumstances. The technique involved growing a crystal of C2, taking an X-ray picture of the crystal and then processing the X-ray image through a computer.
To get the crystals to form, Ruoho and his post doctoral colleague, Dr. Yu Liu, needed to modify the C2 Tang had produced for his studies. Hurley and his post doctoral associate, Dr. Gongyi Zhang, further coaxed the crystals to form by saturating them with forskolin, a plant based, blood-vessel dilating compound that stimulates AC to act without G-protein involvement.
"The crystallography showed that the AC structure looks like a Christmas wreath with two boughs intertwined with each other," said Hurley. "The forskolin is like the Elmer's glue that holds the wreath together."
Knowing the crystal structure of the catalytic core and characteristics of the forskolin binding site will help in the search for a possible natural glue that cements the two wreaths, said the researchers.
Most diseases associated with the cell signaling cycle appear to be related to the receptor or G-protein, but some studies have shown that abnormal levels of adenylyl cyclase may be linked to Alzheimer's Disease, some neuropsychiatric disorders and diseases characterized by uncontrolled cell growth. Understanding AC's structure should help aid drug discovery programs in academia and industry to discover how to regulate AC activity.