Recovery of histamine H3 receptor activity lost in yeast cells through error-prone PCR and in vivo selection

Abstract

Abstract G protein-coupled receptors (GPCRs) are the largest protein family in humans and are drug targets. Yeast, especially Saccharomyces cerevisiae, is a useful host for modifying the function and stability of GPCRs through protein engineering, which is advantageous over mammalian cells. When GPCRs are expressed in yeast, their function is often impaired. In this study, we performed random mutagenesis using error-prone PCR and subsequent in vivo screening to obtain mutants that recovered the activity of the human histamine H3 receptor (H3R), which lost signaling function when expressed in yeast. After screening, four mutations were identified as having recovered activity. Three of these were located near the DRY and NPxxY motifs of the H3R, which are important for activation and commonly found in class A GPCRs. These mutants responded exclusively to the yeast YB1 strain harboring Gi-chimera proteins, showing retention of G protein specificity. Analysis of one of these mutants with recovered activity, C415R, revealed that it maintained its ligand-binding characteristics. The mutations identified in this study may recover the activity of other GPCRs that do not function in S. cerevisiae, and may also be useful in creating mutants, such as stabilized GPCRs in their active conformations.