REEPs, an ER adapter protein family, have differential effects on GPCR expression

Abstract

IntroductionREEPs are members of a growing class of proteins termed ER morphogens or adapter proteins. REEP1 mutations have been implicated in Hereditary Spastic Paraplegia (HSP). To further understand the role of REEPs in ER membrane trafficking and HSP, we have examined their role using two model proteins, alpha2A and alpha2C adrenergic receptors (ARs).MethodsTransiently transfected HEK293 cells expressing individual Flag‐REEP proteins and HA‐alpha2A&C ARs were examined by FACS analysis to determine REEP effects on receptor expression, correlated with ligand binding and deglycosylation analysis.ResultsAnalysis of individual cells expressing both REEPs and ARs revealed a complex effect. Co‐expression of alpha2A ARs with REEP1, REEP2, and REEP6 caused a small increase in plasma membrane AR expression (1.0×, 1.0×, and 0.5× respectively), however there was a larger increase in total AR expression within the cell (1.8×, 1.8×, and 1.2× respectively). A similar analysis of alpha2C AR expression showed a larger REEP effect on surface expression (1.3×, 1.4×, and 1.9× respectively) coupled with a smaller increase in total AR expression (1.2×, 1.2×, and 1.5× respectively). Ligand binding of total membranes showed a similar pattern. Lastly, deglycosylation analysis of ARs revealed that co‐expression of REEPs led to an increase in a minimally glycosylated form of alpha2C ARs.ConclusionsREEPs were originally identified by their ability to enhance GPCR surface expression; however, their effects cannot be generalized. REEPs appear to enhance the production or stability of a minimally glycosylated alpha2C AR. Lastly, co‐IP analysis has revealed that REEPs interact preferentially with alpha2C, but not alpha2A ARs.