Successful expression of a functional yeast G-protein-coupled receptor (Ste2) in mammalian cells.
G-protein-coupled receptors (GPCRs) are membrane-embedded cell signaling devices transducing ligand binding to activation of heterotrimeric G-proteins, providing a paradigm for signaling for yeast and mammals alike. Probing the extent to which yeast GPCRs may couple to mammalian G-proteins has been problematic. In the current work, we explored conditions that enable the cell-surface expression of a yeast alpha-factor pheromone receptor (Ste2). When expressed in human HEK293 cells, Ste2 is shown to bind its ligand alpha-factor, to be functional and catalyze activation of the mitogen-activated protein kinase cascade, and to demonstrate agonist-induced internalization. In response to agonist Ste2 as maintained intracellularly for several hours and avoids the degradation process observed for Ste2 in yeast cells. This is the first successful demonstration of the ability to express a functional yeast GPCR in mammalian cells.
Yin
Dezhong
D
Department of Molecular Pharmacology, University Medical Center, SUNY/Stony Brook, Stony Brook, NY 11794-8651, USA.
Gavi
Shai
S
Shumay
Elena
E
Duell
Ken
K
Konopka
James B
JB
Malbon
Craig C
CC
Wang
Hsien-Yu
HY
D000818:Animals; D001671:Biochemistry / Q000379:methods*; D016466:CHO Cells; D002384:Catalysis; D002460:Cell Line; D006224:Cricetinae; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D008024:Ligands; D020935:MAP Kinase Signaling System; D018613:Microscopy, Confocal; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D018343:Receptors, Adrenergic, beta-2 / Q000378:metabolism; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism; D051964:Receptors, Mating Factor; D018000:Receptors, Peptide / Q000096:biosynthesis*; D015398:Signal Transduction; D013997:Time Factors; D014157:Transcription Factors / Q000096:biosynthesis*
D008024:Ligands; D018343:Receptors, Adrenergic, beta-2; D043562:Receptors, G-Protein-Coupled; D051964:Receptors, Mating Factor; D018000:Receptors, Peptide; D014157:Transcription Factors; D049452:Green Fluorescent Proteins; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3