Mechanical GPCR activation: how to squeeze and stretch a cell into action

It all started with a question

It started with an innocuous question. Mette Rosenkilde, working in the Department of Biomedical Sciences, was studying a GPCR subfamily known as adhesion GPCRs. The adhesion GPCRs were proving particularly difficult to activate. Recently, research had begun pointing to the importance of the mechanical activation of GPCRs and Mette had begun to wonder if this was the problem she faced. Mette decided to reach out to Karen Martinez, a biophysicist and nano-scientist based at the Department of Chemistry. Karen was an expert in GPCRs and the use of nanotools for the development of novel cell assays with novel detection methods. Perhaps Karen could build some kind of nano-device to mechanically activate them? 

Not surprisingly, there was no ready-made solution that Karen could provide to solve Mette’s problem. But the seed had been planted and over the next few years, Karen became increasingly interested in the mechanical activation of GPCRs. Eventually, Karen came across an article which suggested that post-translational modifications play an important role in mechanical activation. She sent the article to Mette. By serendipity, Mette was now collaborating with Christoffer Knak Goth, who was also based at the Department of Biomedical Sciences. His specialty: post-translational modifications (PTMs) of GPCRs. And just like that, the foundations of the present research project were laid.

Mapping out an integrated approach

Mette put Karen and Christoffer in touch and Karen also started discussing the project with Poul Martin Bendix, a biophysicist from the Niels Bohr Institute and expert in membrane biophysics and optical biomanipulation. Between the four of them they started sketching out the scope of the project. Rather than develop a device to activate a particularly difficult subfamily of GPCRs whose mechanosensitive status was unknown, the group will focus on GPCRs whose mechanical sensitivity is already established. Karen and Poul Martin will apply various forms of pressure and stress to cells in the hopes of triggering mechanical activation. To do this, Poul Martin will use optical tweezers, a laser beam that can hold, move and in this case squeeze cells. Meanwhile, Karen will develop nano-devices that can stretch and shrink cells. Both of these experimental approaches are designed to mimic the stresses a cell undergoes in the body, as for example occurs when changes in blood flow cause the cell membrane to expand and contract. Through these tests, the research will establish whether it is these processes that are the final trigger for mechanical GPCR activation.

In addition, the cells and GPCRs that Karen and Poul Martin work on will be analyzed and prepared by Christoffer and Mette so that different cells and samples possess different post-translational modifications. Although it is likely that most GPCRs would have undergone some kind of post-translational modification, they are difficult to detect and their study has been largely neglected. However, recent technological progress is starting to break down this barrier and Christoffer and Mette will use different tools such as in silico analysis, enzymes, inhibitors, mutations and genetically engineered cells to determine the status of the different modifications – and to control them. In this way, the research will explore which – if any – post-translational modification enables mechanical activation. In addition, Poul Martin will study the interactions between mechanical and ligand-activation, to try and understand when, where and why one method of activation becomes more important than the other and how the two triggers influence each other. 

The funding challenge

Finding funding for an interdisciplinary project is notoriously difficult. While everyone recognizes their importance, they tend to perform poorly when sent out for evaluation. At one end of the spectrum, lead investigators often lack – or are perceived to lack - the required expertise in all of the involved disciplines. At the other end, the experts who judge these proposals are themselves specialists and often find it difficult to evaluate a project with ambitions beyond their area of expertise.

To overcome these handicaps, numerous funding bodies have started introducing dedicated interdisciplinary funding instruments. As Karen emphasizes, ‘this is really important because when you mix people from different environments, you force people to get out the box and then you have new ideas popping up.’ And this is exactly what has happened. In 2020, Karen, Mette, Poul Martin and Christoffer were awarded a Novo Nordisk Foundation Exploratory Interdisciplinary Synergy Grant: a DKK 5 million, 2-year grant that allows researchers to develop interdisciplinary ideas and gather some supporting data for their hypothesis.

The journey so far

The grant has allowed each group to hire a dedicated postdoc to the project. At BMI, this includes Jarkko Lackman from Finland who has previous experience in both GPCRs and post-translational modifications. At NBI, Poul Martin has been joined by postdoc Mohammad Arastoo and at CHEM, Karen has hired postdoc André Dias.

Working within their own groups, each collaborating researcher has been busy in the set-up phases ensuring their own piece of the puzzle is ready. At BMI, Christoffer, Mette and Jarkko have been investigating the presence and impact of post-translational modifications on classical signalling. Meanwhile at CHEM, Karen has developed the needed nano-devices. And at NBI, Poul Martin and Mohammad have been working on characterizing the receptors and their organization in plasma membranes. With each separate piece now ready, the team has now started to combine their tools with samples and data expected to start flowing between the labs freely in the beginning of 2022.

Integrated and interdisciplinary

Karen, Mette, Christoffer and Poul Martin’s journey represents a truly integrated and interdisciplinary approach to structural biology. It not only involves the integration of various techniques to study biological structures but it also involves the integration of that structural data with cell biology. Through this combination, pressing research questions have been made accessible by out-of-the-box thinking and seemingly insurmountable challenges have led to the development of novel technological break-throughs.

 

ISBUC will be following their research project over the next year and will report back regularly. In the meantime, if you have any questions or would like to find out more about the project, please contact Karen Martinez: martinez@chem.ku.dk