pH regulation of cell surface receptors

摘要

ABSTRACT Understanding proton-gating (H+-gating) of GPCR activity is a major new frontier in signaling biology. GPCRs regularly encounter coincident H+ signals in a variety of contexts, such as endosomes, tumors, synapses, inflamed tissue, and immune responses. However, the extent to which these signals regulate GPCR function represents a significant gap in our understanding of cellular sensing, communication, and drug discovery. The goal of our research program is to provide this insight for much of the GPCRome. A major goal is the development of pH-intelligent nanobodies that enable the direct tracking and regulation of GPCR function in normal and acidified conditions, with a longer-term plan is to develop the most promising of these precision tools into therapeutics. We have had much success in these efforts. We have successfully engineered the necessary nanobody discovery platform and validated it using known GPCR-nanobody pairs. We have also computationally designed and manufactured new synthetic nanobody libraries which we have begun to screen against pools of diverse GPCRs. Because of our large library sizes and high hit rates, we have discovered a bottleneck in the plating, cataloging, picking, and characterization of yeast colonies carrying putative nanobody hits. The instrument we are requesting, a Mini Qpix small footprint microbial colony picker, will overcome this bottleneck and enable us to expand our processing from hundreds to thousands of nanobody hits. Such capabilities would greatly accelerate the discovery of nanobody regulators of GPCRs and have the potential to illuminate nanobody diversity at a depth and scale necessary for training artificial intelligence algorithms for de novo nanobody design. As such, NIGMS support for our instrument request would catalyze a substantial leap forward in the field.