Mutagenesis of potential transmembrane palmitoylation sites reduces the surface expression of a chimeric membrane receptor

Abstract

A chimeric receptor with an alpha7 nicotinic receptor extracellular segment & serotonin type 3A receptor transmembrane (TM) & cytosolic (CY) segments, when transiently transfected into human tsA201 cells, is highly palmitoylated, which appears to promote cell‐surface & functional expression (Drisdel et al. 2004). The TM & CY segments contain 5 potential palmitoylation sites: C231 & C278 in TM domains M1 and M3, respectively, & C322, C344 & C357 in the M3–M4 CY loop. Cys‐to‐ala mutants were transiently expressed in tsA201 cells & the number of surface & intracellular receptors & surface receptor function were assessed. Surface expression of receptors with the double TM segment mutation C231A/C278A was only 48% that of wild‐type (WT) receptors (p=0.01), while expression of receptors with the triple CY loop mutation C322A/C344A/C357A did not differ significantly from WT. Expression of C278A was 81% of WT (p=0.02) while expression of C231A did not differ significantly from WT. Mutants C278A/C322A/C344A/C357A & C231A/C278A/C322A/C344A/C357A both showed significantly lower surface expression than WT (47%, p=0.005 & 29%, p=0.05, respectively). The amount of intracellular receptors produced in cells transfected with the above TM segment mutants did not differ significantly from that of WT, suggesting that removal of sulfhydryl groups in the TM segments interfered with the trafficking of fully‐assembled receptors to the cell surface. Cells expressing the 5‐cysteine mutant receptor accumulated 2‐fold higher intracellular calcium levels than cells expressing WT (p<0.001). [NINDS/SNRPMI 5U54NS39408 (VAE); T32 DA07255 (JA); NS043782, DA13602, NS043782, & DA019695 (WNG); NIGMS/MBRS 2SO6GM50695 (RH)]