Debilitating lupus disease may have simple cause, new UCSF research shows

December 20, 2000

Scientists have discovered that a single defect in a common protein causes a severe auto-immune disease resembling lupus - the debilitating, poorly understood malady that affects 100,000 people in the U.S., mostly young women. The discovery points to a new drug target to treat a range of auto-immune diseases. The research by scientists at the University of California, San Francisco combines genetic experiments in mice with studies of protein interactions.

Drawing on a refined understanding of how a wedge of atoms can convert molecular partners into antagonists, the new finding provides subtle but powerful evidence that lupus in humans might sometimes be an inherited disease with a simple genetic root, rather than an accumulation of a number of genetic defects as many have thought.

The study provides new evidence that a single genetic mutation can cause auto-immune disease, and also offers rare, compelling evidence that such a defect is dominant over the normal condition, making the genetic change hundreds to thousands of times more likely to cause disease than if it were a recessive trait. If a gene for a disease trait is dominant, a single copy of the gene can cause the malady, whereas if it is recessive, copies from both parents are required. Most previously identified genetic causes of auto-immune disease are recessive.

The finding also supports the view that what is called lupus may in fact be a collection of a number of different diseases with a common pathway, the scientists suggest. Their research showed that a lupus-like syndrome could result from a single amino acid change in a key protein on the surface of cells of the immune system. Known as a transmembrane receptor protein, this molecule normally receives signals from outside the cell and triggers internal responses. Since the protein is central to nearly all immune cells in humans as well as mice, the finding suggests that the range of symptoms associated with lupus - inflammation of heart and lungs, kidney failure, arthritis, and other forms of auto-immunity - could all derive from the single defect in the signaling pathway protein.

"Lupus is a devastating disease that affects one in every 2,000 people in the U.S. - and attacks nine females for every male," said Arthur Weiss, MD, PhD, Howard Hughes Medical Institute Investigator and UCSF professor of medicine.

"But while researchers have studied the disease intensively, the cause -- or causes -- have proven difficult to pin down. It may well be that at least part of the answer to the puzzle lies with a very small change in a signaling protein that underlies many different cell functions," he said.

Weiss is senior author on the paper describing the research in the December 22 issue of the journal Cell. Lead author is Ravindra Majeti, PhD, an MD/PhD candidate at UCSF working with Weiss.

The discovery comes from detailed studies of the transmembrane protein straddling the worlds inside and outside of lymphocytes and other cells of the immune system. These receptor proteins are like spokes piercing the cell membrane. Part of the receptor pokes into the cell and part extends out from the cell surface. The external "domain" receives signals from outside the cell which regulate responses inside -- ranging from increasing the cell's glucose supply to starting a new cell division cycle to generate more lymphocytes.

The research examined a natural chemical process known as dimerization, in which neighboring receptors on the cell surface become physically paired. Scientists have known for a decade or more that this pairing of two receptors by an incoming signal or ligand activates most types of receptors, triggering the internal cellular response through a process known as phosphorylation. This insight came from work on a receptor family known as receptor tyrosine kinases (RTKs).

Weiss and Majeti focused on a receptor protein called CD45 in a class known as receptor protein tyrosine phosphatases or RPTPs. They function in a manner opposite to RTKs. Rather than adding phosphate molecules - the process known as phosphorylation - RPTPs take away phosphate molecules - dephosphorylation - inside the cell.

In earlier research Weiss and colleagues had experimentally dimerized CD45 protein receptors and found that they responded in an unexpected way. Rather than becoming activated by the pairing of separate CD45 molecules, the receptors' signaling capacity was shut off.

"All previous work in the signaling field had suggested that dimerizing cell surface receptors activates their signaling function," said Weiss. "So, we were quite surprised to find that dimerization of CD45 inhibited its function."

This unusual finding suggested to Weiss that dimerization of CD45 may play a critical, and unrecognized regulatory role. All mammals, from mice to humans, recruit cells of the immune system to fight invading pathogens such as viruses and bacteria, but they must have some way to shut the process down or else they risk falling prey to uncontrolled immune cell activation leading to auto-immunity, severe inflammatory disease and destruction of organs.

Dimerization of CD45, Weiss hypothesized, may be the chemical switch mammals use to shut off the normal immune response, thereby averting the kinds of immune dysfunction caused by uncontrolled immune activation found in lupus and many other immune diseases such as rheumatoid arthritis and multiple sclerosis.

Weiss and Majeti had other important clues that allowed them to test this hypothesis. A few years ago other researchers used the technique of x-ray crystallography to examine the structure of the interior cellular domain of a related RPTP protein. They found that these intracellular domains formed dimers in the crystal. The crystal revealed that a wedge-like projection on each partner in the pair of receptor molecules blocked the active chemical site of the other. The two, in other words, were locked in a paralyzing embrace.

Crystallography also revealed precisely what amino acids make up the wedge in the protein. "We also knew that those amino acid sequences were conserved in related receptor proteins, including CD45," said Majeti, "so we hypothesized that CD45 regulates lymphocyte activation through the action of the wedge when pairs of these CD45 receptor proteins are dimerized."

Weiss and Majeti tested their hypothesis by mutating the portion of the CD45 gene in mice that codes for the wedge feature. They created a mouse that differed from its parents by only a single amino acid, but that amino acid change meant that their CD45 proteins lacked the wedge function. They predicted that if dimerization of CD45 inactivated its signaling function, the CD45 molecules in the mutant mice should no longer be subject to this form of regulation.

Sure enough, the animals' immune cells proliferated out of control, triggering the auto-immune disease that has the features of human lupus, including severe kidney inflammation.

The experiments confirmed that the dimerization shuts off receptor function and that this is accomplished through the action of the wedge portion of the protein in the CD45 cytoplasmic domain. Although the researchers expect that the CD45 receptors normally pair up in this fashion, how this is controlled is not yet clear.

Many experiments have established that mice are a useful model for a number of immune diseases in humans. Majeti and Weiss now hope to discover that at least a subset of people with lupus or other auto-immune diseases have the genetic mutation that inactivates the CD45 receptor protein regulatory activity, causing the immune dysfunction.

"If we confirm that CD45 plays a regulatory role to control proliferation of T-cells or B-cells," says Majeti "then developing a drug to dimerize CD45 proteins might well provide a treatment for a range of pernicious auto-immune diseases."
-end-
Co-authors on the Cell paper and collaborators in the research are Zheng Xu, PhD candidate in the UCSF cell biology program; Tristan Parslow, MD, PhD, professor of pathology and of microbiology and immunology; Jean Olson, MD, professor of pathology; and Nigel Killeen, PhD, assistant professor of microbiology and immunology, all at UCSF; and David Daikh, MD, PhD, assistant professor of medicine at UCSF and at the Veteran Affairs Medical Center in San Francisco.

The research is funded by the Howard Hughes Medical Institute, the National Institutes of Health and UCSF's Rosalind Russell Center for Medical Research in Arthritis.

University of California - San Francisco

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