Another Job Discovered for a Master Metabolic Off-Switch
DURHAM, N.C. -- Researchers have discovered that an important cellular "off-switch" that desensitizes receptors on the cell surface also regulates a second deactivation mechanism that had not been suspected before. Their finding that the off-switch, known as beta-arrestin, operates in two distinct ways hints at a broader set of regulatory roles for the molecule, said the scientists.
Receptors are proteins that nestle in the cell membrane, and which translate external chemical signals such as hormones into a cellular response. Discovering the detailed mechanisms of beta-arrestin action is important because the receptors it "downregulates" number perhaps in the
thousands, and control processes throughout the body -- including heart rate; blood pressure; endocrine function; the senses of sight, smell and taste; pain tolerance, and the euphoric high of addictive drugs. Thus, new basic knowledge about the action of beta-arrestin could have implications for development of drugs for a wide array of disorders.
In an article in the October 25, 2002, "Science," Howard Hughes Medical Institute investigator Robert Lefkowitz, M.D., at Duke University Medical Center and his colleagues reported their studies of the role of beta-arrestin in regulating a "second-messenger" molecule called cyclic AMP (cAMP).
This molecule launches a cascade of metabolic signals into the cell when it is produced by activation of receptors such as the "G-protein-coupled receptors" (GPCRs) studied by Lefkowitz's laboratory. The role of beta-arrestin is to desensitize GPCRs by regulating cAMP levels, to prevent over stimulation of the cell.
"It's been known for a number of years that there are two major mechanisms by which the levels of cyclic AMP are regulated," said Lefkowitz. "One is the rate at which cAMP is synthesized by an enzyme called adenylate cyclase, which in turn is activated by receptors. And the other mechanism is the rate of cyclic AMP degradation, which is carried out by a large family of enzymes called phosphodiesterases."
Lefkowitz and his colleagues had discovered in previous studies that beta-arrestin desensitizes receptors by binding directly to them, blocking the machinery required to activate adenylate cyclase.
"However, we had absolutely no idea that beta-arrestin might also act on phosphodiesterases to help them degrade cyclic AMP." said Lefkowitz. "All the textbooks say there's no relationship between these two mechanisms."
However, said Lefkowitz, other scientists had published experimental results hinting that levels of phosphodiesterases (PDEs) were not uniform throughout the cell, and that the enzyme might be moving to places where it is needed. Also, said Lefkowitz, a realization was dawning that beta-arrestin molecules played a broader role in cell processes than previously believed.
"Over the past five years in my laboratory, we have been learning that beta-arrestins are not just molecules that get pulled to the receptors to desensitize them; but increasingly it is clear that they serve all sorts of other functions as scaffolds and adapters which bind to proteins to
promote signaling through different pathways."
Thus, the lead scientists on the "Science" paper -- Stephen Perry, George Baillie and Trudy Kohout -- set out to explore whether beta-arrestin might possess yet a second role in desensitizing receptors -- by recruiting PDEs to the membrane to degrade cAMP.
In their initial experiment, using chemicals that switched on receptors, the scientists traced the movement of the PDE enzymes in cultured cells. They found that in such cells the enzymes did move from the cell's interior to its membrane. Also, further studies confirmed that the enzymes did, indeed, attach themselves to the receptors.
To discover the role of beta-arrestin, the scientists tried the same experiment in cells engineered to lack beta-arrestin -- which occurs in two forms, beta-arrestin1 and beta-arrestin2. In those experiments, they found that knocking out either or both beta-arrestin forms stopped recruitment of PDEs to the membrane. Test tube studies also confirmed that beta-arrestin and PDE bound to one another very tightly.
"We could have stopped there, but we really wanted to demonstrate that this mechanism, which is totally novel, was of some functional significance," said Lefkowitz. "So, we devised an experiment in which we produced a form of PDE that was catalytically dead, but which could compete with and block normal PDE at the membrane. When we added this altered PDE to the cell cultures, we discovered that it blocked recruitment. And our measurements showed that the receptors remained active when stimulated.
"What these experiments tell us is that these two mechanisms for regulating the levels of the second messenger, cyclic AMP, at the membrane -- which we had viewed as two completely separate and unrelated mechanisms -- are really two aspects of the same thing," said Lefkowitz. "They are completely coordinated." According to Lefkowitz, the finding emphasizes the
intricate synergy of the action of beta-arrestin.
"It is an elegant mechanism from a design point of view," he said. "It makes a lot of sense biologically, and provides insight into how beautifully coordinated these systems are. It also points out once again how little we know, since for years we felt confident that these were two very distinct mechanisms of regulation." Lefkowitz emphasizes that much more remains to be discovered about beta-arrestin.
"Work in our lab and others is clearly indicating that the beta-arrestins are master regulators, serving not just as molecules to turn off a vast array of receptors, but to turn them on and to link them as adapters and scaffolds to other metabolically important enzyme systems.
"The receptors that we study were originally named G-protein-coupled receptors, but they could just as well have been called arrestin-coupled receptors," said Lefkowitz, who himself has adopted the broader label "seven-membrane-spanning receptors." The scientists' work is also supported by the National Institutes of Health.