G-protein coupled receptors (GPCRs) comprise the largest family of transmembrane receptors, serving to transfer cues from the extracellular space into eukaryotic cells. This relay of information is often mediated by the binding of extracellular ligands to these receptors, which in turn initiate intracellular signaling cascades via associated G-proteins. In addition to constituting approximately 1% of mammalian genomes, members of the GPCR superfamily also represent the targets for more than 50% of all pharmaceutical agents. Despite their critical biological and clinical importance, the only high-resolution structure available is that of bovine rhodopsin, which, unlike most GPCRs, is naturally abundant. Most naturally or heterologously expressed GPCRs do not yield quantities necessary for performing detailed biophysical studies, so protein expression is currently a major bottleneck in obtaining three-dimensional structures. An attractive idea for increasing the expression level of a GPCR (and possibly improving its biophysical properties) is to perform directed evolution on the sequence. Here, we present a novel directed evolution approach for the engineering of integral membrane proteins such as GPCRs. We have used this platform to increase the heterologous expression level of a GPCR and also to alter its ligand-binding selectivity. The nature of the mutations that arose from these screens will be discussed in the context of GPCR folding and function.

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