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Structure Of Key Regulatory Enzyme Reveals It To Be A Molecular 'Octopus'

Date:
June 20, 2003
Source:
Duke University
Summary:
After seven years of work, researchers have succeeded in deducing the three-dimensional structure of an elusive and complex protein enzyme that is central to regulating the body's largest family of receptors. These receptors, called G-protein-coupled receptors, nestle in the cell membrane and respond to external chemical signals such as hormones and neurotransmitters, to switch on cell machinery.
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DURHAM, N.C. -- After seven years of work, researchers have succeeded in deducing the three-dimensional structure of an elusive and complex protein enzyme that is central to regulating the body's largest family of receptors. These receptors, called G-protein-coupled receptors, nestle in the cell membrane and respond to external chemical signals such as hormones and neurotransmitters, to switch on cell machinery.

The thousands of such receptors throughout the body play a fundamental role in the mechanisms of sight, smell and taste, and in regulating heart rate, blood pressure and glucose metabolism. The receptors are by far the most common target for drugs that affect cardiac output, blood pressure and many other physiological functions. Thus, said the researchers, their fundamental discovery could guide pharmaceutical companies in creating a new class of drugs that aim not at blocking the receptors themselves, but at modulating the machinery that regulates them. Such drugs could treat a range of disorders from congestive heart failure to Parkinson's disease, they said.

The newly revealed structure of this receptor "off-switch" -- called a G protein-coupled receptor kinase (GRK) -- reveals the protein as the molecular equivalent of a three-armed octopus, with independent segments capable of performing multiple regulatory functions at once. Kinases are enzymes that act as molecular switches by adding phosphates to other proteins.

The researchers -- led by Howard Hughes Medical Institute investigator Robert Lefkowitz at Duke University Medical Center and John Tesmer of the University of Texas at Austin -- reported their findings in the May 23, 2003, issue of the journal Science. The team also included scientists from the University of Texas at Austin and University College London. Also on the research team was Darrell Capel of Duke.

"Fundamental to the regulation of all these receptors is the ability to damp their signaling in the face of constant stimulation," said Lefkowitz. "Years ago, we had discovered that this down-regulation occurs due to a phosphorylation of the activated receptor that triggers binding of a protein called beta arrestin. This protein stops further G protein signaling and acts as an adaptor and scaffolding that connects to other signaling molecules."

Thus, the cellular "stop signal" not only turns off the G protein, but immediately tags the receptor for recycling into the cell interior and turns on other signaling pathways, said Lefkowitz. His laboratory identified that enzyme as GRK, but a central mystery was how the family of GRK enzymes fulfills their intricate regulatory duties.

In the latest work the researchers deduced the structure of GRK2, the member of the GRK enzyme family that is active in heart muscle and many other tissues.

Critical to solving that mystery was obtaining the three-dimensional structure of GRK2 using X-ray crystallography. In this technique, pure crystals of a protein are bombarded by an intense X-ray beam, and the protein structure is deduced by analyzing the pattern of the beam's diffraction. This structural determination was done by co-author John Tesmer and his colleagues.

The resulting structure revealed the details of three regions, or domains, of the GRK2 enzyme, which had earlier been identified by biochemical studies in the Lefkowitz laboratory:

* The central, or catalytic domain is the region that triggers the phosphorylation reaction

* The "regulator of G protein signaling homology" (RH) domain attaches to the G protein to switch it off, and

* The "PH" domain enables GRK2 to home in on the G protein at the cell membrane and attach to it.

To reveal how GRK2 interacts with the G protein, the researchers obtained the structure of GRK2 attached to a subunit of the G protein to which it normally binds, or complexes. Lefkowitz noted that a particularly striking achievement was the production of pure crystals of the highly complicated protein complex by Tesmer and his colleagues.

"The results of this prodigious effort were some really striking and unanticipated insights into the structure of the GRK2 complex," said Lefkowitz. "For one thing, the three domains are not aligned in a straight line, but assembled as if they were the three vertices of an equilateral triangle. And their spacing allows them to perform their docking and catalytic functions simultaneously.

"This means that GRK2 could be a remarkably effective and multitasking mechanism for turning off G protein signaling." Thus, he said, the GRK2 is built to bind to the receptor and phosphorylate it, allowing attachment by beta arrestin, and at the same time, bind the G protein to switch it off.

"This structural determination has significance at two levels," concluded Lefkowitz. "First, it gives us important new information about the basic biology of this important regulatory mechanism. And second, it gives us the detailed molecular coordinates of this structure that guide drug developers in designing specific compounds to regulate the enzyme."


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Materials provided by Duke University. Note: Content may be edited for style and length.


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Duke University. "Structure Of Key Regulatory Enzyme Reveals It To Be A Molecular 'Octopus'." ScienceDaily. ScienceDaily, 20 June 2003. <www.sciencedaily.com/releases/2003/06/030620080237.htm>.
Duke University. (2003, June 20). Structure Of Key Regulatory Enzyme Reveals It To Be A Molecular 'Octopus'. ScienceDaily. Retrieved May 22, 2017 from www.sciencedaily.com/releases/2003/06/030620080237.htm
Duke University. "Structure Of Key Regulatory Enzyme Reveals It To Be A Molecular 'Octopus'." ScienceDaily. www.sciencedaily.com/releases/2003/06/030620080237.htm (accessed May 22, 2017).

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