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Duke s Mark Caron on GPCR Signaling Pathways and Drug Discovery

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At A Glance

Name: Mark Caron

Position: Research professor, department of medicine; and professor of cell biology, Duke University Medical Center; Interim director, Center for Human Disease Models, Institute for Genome Science and Policy, Duke University; Co-founder and chairman of scientific advisory board, Norak Biosciences

Duke University professor Mark Caron has long been interested in the mechanisms and regulation of G protein-coupled receptors, and more recently in the mechanisms of neurotransmission as controlled by neurotransmitter transporters. As a co-founder of biotech firm Norak Biosciences, Caron also helped develop that company’s flagship technology, Transfluor, a fluorescent assay for investigating a wide variety of GPCRs in living cells. Caron took a few moments last week to discuss some important research regarding GPCRs conducted by him and several Duke colleagues and recently published in Science.

How did you become interested in studying GPCR-related signaling pathways?

GPCRs — 7-transmembrane receptors — this is a large family of receptors — probably in the thousands, and certainly between 500 and 1,000, mediating everything from vision to smell to taste, and just about any other signaling molecule in the body. There’s this paradigm: They were all called G-protein coupled receptors in the old days because many of them, if not all of them, signal through activating G-proteins and activating effector molecules, such as adenelyl cyclase, and eliciting second messengers inside the cell. Essentially, in all cells that have GPCRs, there’s a competing reaction — actually, many competing reactions — that turn off the signal, because if you couldn’t turn off the signal, it wouldn’t be very good for biology. One of the very common mechanisms is for one of the activated receptors to become a substrate for what we would have called selective, or specific, receptor kinases called GPCR kinases. These receptors, once phosphorylated, get recognized by the arrestin molecules, and eventually uncouple the ability of those receptors to signal further through the G-protein dependent pathway.

That’s where it really gets interesting. Our colleague Bob Lefkowitz and I have made knockouts of all these kinases and arresting molecules, and when you knock out these proteins, you enhance the responsiveness of various responses in animals. They truly desensitize some signaling. However, back in the mid-1990’s, we had begun to have a change of mind fueled by some results that we got. We began to accumulate evidence from cellular systems that this desensitization machinery — the kinases and the arrestins — not only desensitize receptors, but also play other roles, what we would call pleiotropic roles. The first of these was based on a couple of observations: one made by our colleague Jeff Benovic at Thomas Jefferson [University] — that arrestins interacted with clathrin, proteins that are involved in endocytosis, and another observation that we made: that arrestins appear to be essential for internalizing GPCRs. This is what led to the discovery of the Norak technology — the Transfluor assay — because then we went on to tag the arrestin with GFP, and Larry Barak and my group essentially popularized this assay. There are other ways of internalizing GPCRs once they are activated, but many of them do interact with the arrestin after they get phosphorylated. So the idea would be to stimulate the receptors, couple to the G proteins, listen to the second messenger, and as a competing reaction that occurs quite rapidly thereafter, you get phosphorylation of the receptors and interaction with arrestin. Then the receptor that’s complexed with the arrestin is then internalized through clathrin-coated pits, where it can presumably be dephosphorylated and recycled back to the plasma membrane as a competent receptor. The Norak technology is essentially a visualization of this initial step — the translocation of cytosolic arrestin to an activated recptor.

Then there was another piece of data that came in 1999 [from] Bob Lefkowitz’s lab and my lab: Working together made the observation that a GPCR-arrestin complex could interact with a signaling molecule — namely Src, the tyrosine kinase. That led to the idea that maybe what happened is this complex of phosphorylated receptors bound to a ligand, which we thought was kind of a dead complex, may not be after all. Maybe it is involved in some signaling. Then a lot of evidence from cellular systems — lots of it from Bob Lefkowitz’s lab, and some of it from other labs like Nigel Bunnet’s at UCSF — that indeed, the activated GPCRs, phosphorylated and interacting with arrestin, were able to scaffold various signaling molecules, such as Erk1 and Erk2, p38, Jnk, and things like that. In a sense, the idea came that probably what happened is that there is a complex that may serve as a two-pronged mechanism: You signal through G-proteins and other receptors and elicit a second messenger; and concomitant with that, there’s a more stable signaling wave that activates all kinds of things that were originally associated mainly with tyrosine kinase receptors.

So this is the main thrust of the recent papers?

The work that we just published in Science relates to both of these things. There is also what I call the non-canonical 7-transmembrane signaling system. Hedgehog, patch, and smoothen, that we are now describing, is one of these things, and there’s another system called WIF. These are proteins originally identified in Drosophila that are involved in development called wingless, or WIF for short. They are ligands that are morphogens, and bind to receptors that are called frizzled, which are 7-transmembrane proteins. The same thing is true for smoothen — hedgehog is a signaling molecule that binds to a protein called patch, a 12-transmembrane protein. These things were discovered in Drosophila originally, there are mammalian homologues. The thing that has been interesting is downstream of frizzled and smoothen, these things effect gene transcription. But because many of the people that have studied this have done it in a genetic way, how these signal to the transcription factor has been kind of [unknown.] There has been very scant evidence that these 7-transmembrane domain proteins can activate G-proteins.

Last year, we published in Science that frizzled can interact with arrestin, but probably in a slightly different way. This was the first observation that some of these 7-transmembrane proteins might utilize some of the same molecules that are involved in the desensitization of GPCRs. At the same time, our labs were pursuing the other aspect of this, the smoothen.

What are the cell biology and drug discovery implications of these recent papers?

[Lefkowicz’s group] basically showed that smoothen is capable of interacting with arrestin, and probably mediates the internalization of smoothen once it is activated. The mechanism of how patch inhibits smoothen and how it becomes activated is not totally clear. There are compounds that bind to smoothen that can inhibit it. This really showed that smoothen may well function like a canonical 7-transmembrane receptor — that is, it gets phosphorylated, and it probably gets internalized, and the arrestin might help to internalize it. This has never been shown before.

So what are the implications for drug discovery? If you activate smoothen or you make mutations in this hedgehog pathway, it leads to several forms of cancer — basal cell carcinoma and medulloblastoma, which is a more aggressive brain tumor. People have been looking for assays for compounds that might inhibit smoothen, because if you express it in cells by itself, it’s constitutively active, and we know there are mutants of this. So this assay not only gives the exciting results that smoothen might function like a 7-transmembrane protein, but it provides an assay where you can just put smoothen into a cell, look at the translocation of arrestin, and use that to find ligands — sort of like how we find ligands at Norak for any GPCR.

So you also showed similar results using zebrafish assays?

Yes, we can also do this in zebrafish. These are live animals. And basically, the bottom line of this second study is that maybe arrestin 2 may be implicated with smoothen, but it may be a signaling component of the smoothen pathway.

What are the relative advantages of using the zebrafish assays?

The advantage of zebrafish is that you can mutate them, look at lots of them, and they are transparent, so you can see some of the phenotypes very early. Instead of a mouse, where it takes a year to make a knockout, you can have the results here much faster. The other advantage is that we’ve now used this to look at mutants of the arrestin … basically as an assay. The zebrafish becomes our test tube, essentially, for activities of various mutants and things like that, and it could probably be applicable for finding drugs for these interactions. One of the difficult things in the drug industry is finding drugs that target protein-protein interaction, and this might be an interesting avenue to explore.

You were involved in the invention of Transfluor… what are the major advantages of this assay in drug discovery applications?

The main advantage is that it’s one assay that’s essentially applicable to many, many GPCRs. Not all of them — you have to work the conditions — but certainly it’s very versatile. So far it seems to be a very robust assay.

Did you use the Norak technology for these assays, as well?

We have clones of zebrafish arrestin and smoothen, and they do exactly the same thing when you put them in cells that mammalian arrestin and smoothen do. But we didn’t use that per se. We used a chemical way to tone down the message of the arrestin — they’re essentially stable analogs of DNA called morpholinos. We inject the morpholinos that are specific for inhibiting the translation of arrestin at the one-cell stage, and then we look at the development, and if the arrestin is important for development, then you will have mutations, and the developing embryo will look different. We discovered this totally independent of the other types of studies that we were doing. We were actually doing something else with the zebrafish, and stumbled upon this. So when we downregulated the arrestin, we basically found that it replicated all of the phenotypes that one observes if you knock out hedgehog or smoothen. Then we were able to show that we could essentially rescue this, so if we activate the pathway downstream of smoothen, we can rescue the defective phenotype in development. So we use an epistasis experiment to essentially convince ourselves that the arrestin probably does interact at the level of smoothen, and probably because of the effect that we get, it’s probably functioning either as a positive regulator, or mediator of smoothen. So the question that’s left is: What is the mechanism?

So the next step for your research group is trying to characterize this mechanism?

We were interested in trying to understand the mechanism by which the interaction between smoothen and arrestin — if indeed it does translocate a message — works. The other thing is: If smoothen interacts with arrestin because it’s phosphorylated with a kinase, we would like to know which ones are involved. Quite interestingly, there are several of these kinases expressed in the zebrafish genome, so we’re actually looking at some of them.

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