Modifying Expression Modes of Human Neurotensin Receptor Type 1 Alters Sensing Capabilities for Agonists in Yeast Signaling Biosensor

Abstract

Neurotensin receptor type 1 (NTSR1), a member of the G-protein-coupled receptor (GPCR) family, is naturally activated by binding of a neurotensin peptide, leading to a variety of physiological effects. The budding yeast Saccharomyces cerevisiae is a proven host organism for assaying the agonistic activation of human GPCRs. Previous studies showed that yeast cells can functionally express human NTSR1 receptor, permitting the detection of neurotensin-promoted signaling using a ZsGreen fluorescent reporter gene. However, the fluorescence intensity (sensitivity) of NTSR1-expressing yeast cells is low compared to that of yeast cells expressing other human GPCRs (e.g., human somatostatin receptors). The present study sought to increase the sensitivity of the NTSR1-expressing yeast for use as a fluorescent biosensor, including modification of the expression of human NTSR1 in yeast. Changes in the transcription, translation, and transport of the receptor are attempted by altering the promoter, consensus Kozak-like sequence, and secretion signal sequences of the NTSR1-encoding gene. The resulting yeast cells exhibited increased sensitivity to exogenously added peptide. The cells are further engineered by using cell-surface display technology to ensure that the agonistic peptides are secreted and tethered to the yeast cell wall, yielding cells with enhanced NTSR1 activation. This yeast biosensor holds promise for the identification of agonists to treat NTSR1-related diseases.

Coupling of Human Rhodopsin to a Yeast Signaling Pathway Enables Characterization of Mutations Associated with Retinal Disease

“…Mirroring our mammalian cell-based observations, P23H and M39R were poorly distributed across the yeast plasma membrane, with highly localized "patchy" expression, while G51A localized consistently to the membrane (Figure 5A). The peri-nuclear ring observed is similar to the localization of other human GPCRs expressed in yeast, indicating the ER membrane (O'Malley et al 2009;Hashi et al 2018). Although P23H was observed to form aggregates in the ER of our mammalian cells, this was not observed in yeast; however, M39R did appear to form aggregates in the cytosol of yeast.…”

Comparative analyses of site‐directed mutagenesis of human melatonin MTNR1A and MTNR1B receptors using a yeast fluorescent biosensor

“…DNA fragments containing the mutated MTNR1A gene (without a stop codon) were PCR‐amplified from pGK421‐MTNR1A‐xxx (xxx: D73N, C100A, N124A, R125A, Y126A, C177A, V192T, H195A, V192T+H195A, or P253A, respectively) using primers #1 and #45. The resulting amplicons were digested with Nhe I + Sal I, then ligated between the PGK1 promoter ( P PGK1 ) and the mUkG1 gene in a manner similar to pGK421‐NTSR1‐mUkG constructed previously (Hashi et al, 2018), yielding the plasmids pGK421‐MTNR1A‐xxx‐mUkG (xxx: D73N, C100A, N124A, R125A, Y126A, C177A, V192T, H195A, V192T+H195A, or P253A, respectively). The plasmids pGK421‐MTNR1B‐xxx‐mUkG (xxx: D86N, C113A, N137A, R138A, Y139A, C190A, V205T, H208A, V205T+H208A, or P266A, respectively) were similarly constructed with primers #3 and #46.…”

“…R125A, Y126A, C177A, V192T, H195A, V192T+H195A, or P253A, respectively) using primers #1 and #45. The resulting amplicons were digested with NheI + SalI, then ligated between the PGK1 promoter (P PGK1 ) and the mUkG1 gene in a manner similar to pGK421-NTSR1-mUkG constructed previously (Hashi et al, 2018), yielding the plas-…”

Biosensing Techniques in Yeast: G-Protein Signaling and Protein-Protein Interaction Assays for Monitoring Ligand Stimulation and Oligomer Formation of Heterologous GPCRs

“…EGFP genes on the genomes of IMFD-70 and IMFD-72 were replaced with ZsGreen genes to generate IMFD-70ZsD and IMFD-72ZsD strains, resulting in the drastic improvement in bright fluorescence and high S/N ratio in the NTSR1 signaling assay [70]. Recently, Hashi et al modified the expression modes of the human NTSR1 receptor by altering the promoter, consensus Kozak-like sequence, and secretion signal sequences of the receptor-encoding gene [83]. The resulting yeast cells exhibited increased sensitivity to exogenously added neurotensin [83].…”

“…Recently, Hashi et al modified the expression modes of the human NTSR1 receptor by altering the promoter, consensus Kozak-like sequence, and secretion signal sequences of the receptor-encoding gene [83]. The resulting yeast cells exhibited increased sensitivity to exogenously added neurotensin [83].…”