Scientists Use Scalable Multiplexing Technology to Study Clinically Relevant GPCR Interactions
By Chris Haake
In a conference presentation, proteomics expert Jochen Schwenk describes his team’s use of xMAP® Technology for studying GPCR-RAMP interactions and expression levels
At this past year’s Drug Discovery & Development virtual event, hosted by Labroots, attendees were treated to a fascinating talk about analyzing G protein-coupled receptors (GPCRs) by Jochen Schwenk, a professor of translational proteomics at KTH Royal Institute of Technology in Sweden and a leader with the Human Protein Atlas.
As Schwenk noted, GPCRs are both extremely important for human health and exceedingly difficult to study. With more than 800 related receptors, GPCRs represent a very large family of membrane proteins that are targeted by many existing therapies. These proteins have been linked to roles in immune response, signaling, sensory perception, neurotransmission, and more. There seems to be quite a bit of remaining potential in this family, Schwenk noted, as more than half of these proteins have never been explored as drug targets.
However, all that promise means little if scientists cannot characterize the proteins accurately.
Among the many challenges Schwenk noted:
- GPCRs are challenging to research because they are embedded in a lipid membrane, where many tools cannot reliably access them
- GPCRs are highly sensitive to environmental changes; once removed from the membrane, they can unfold and make it impossible to study their natural binding patterns
- GPCRs are quite homologous, so any technology to interrogate them must be specific enough to differentiate an individual GPCR from all the others
- Because there are so many GPCRs, scientists need a scalable approach to study them, but traditional cell-based assays typically cannot query the hundreds or even thousands of interactions relevant to these proteins
What’s needed, Schwenk proposed, is a versatile and flexible testing system. Fortunately, his extensive proteomics expertise includes more than two decades of work with Luminex’s xMAP® Technology, a bead-based multiplexing system with strong sensitivity and specificity. Because all reactions occur in solution, rather than on a plate or other fixed substrate, GPCRs can maintain their natural shape for more reliable downstream results.
Schwenk described projects for which he and his team deployed xMAP Technology to learn more about how GPCRs interact with another group of proteins (receptor-activity modifying proteins, or RAMPs) that can be influential in altering GPCR function. Using epitope tags with libraries of GPCRs and RAMPs, they were able to measure receptor expression, identify the presentation of epitopes, and spot interactions with RAMPs — all from the same workflow.
Their work was initially published as a pilot project analyzing 25 secretine-like GPCRs using dual-epitope-tagged constructs, and was more recently expanded to cover more than 200 GPCRs in efforts that also confirmed the specificity of more than 400 antibodies. Interestingly, they found that about 60% of antibodies tested showed only on-target binding, a higher percentage than expected based on past analyses. Schwenk posited that the difference stems from how well GPCRs behave in solution compared to traditional physical substrates. More than a quarter of antibodies showed no target activity at all.
In one illustration of how these results matter for specific clinical indications, Schwenk and his team studied the knock-on effects of GPCR-RAMP binding for a receptor associated with cholestatic itch in liver disease. They demonstrate how xMAP Technology allowed them to identify the specific RAMP interacting with the receptor to change expression levels. In addition, they turned to the deep learning AlphaFold tool to predict the structure of the receptor-RAMP complex.
A positive outcome of all this work, Schwenk noted, is that Luminex’s multiplexing technology has made it possible for his team to study a very challenging and clinically relevant class of membrane receptors and to reveal key interactions that play an important role in downstream biology. The scalability of the platform lends itself well to a family as large as GPCRs and supports the interrogation of many different characteristics, such as expression levels and antibody binding.
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