Flow cytometric analysis of the calcitonin receptor-like receptor domains responsible for cell-surface translocation of receptor activity-modifying proteins

Abstract

The three receptor activity-modifying proteins (RAMPs1, -2, and -3) associate with a wide variety of G protein-coupled receptors (GPCRs), including calcitonin receptor-like receptor (CRLR). In this study, we used flow cytometry to measure RAMP translocation to the cell surface as a marker of RAMP-receptor interaction. Because VPAC2 does not interact with RAMPs, although, like CRLR, it is a Family B peptide hormone receptor, we constructed a set of chimeric CRLR/VPAC2 receptors to evaluate the trafficking interactions between CRLR domains and each RAMP. We found that CRLR regions extending from transmembrane domain 1 (TM1) through TM5 are necessary and sufficient for the transport of RAMPs to the plasma membrane. In addition, the extracellular N-terminal domain of CRLR, its 3rd intracellular loop and/or TM6 were also important for the cell-surface translocation of RAMP2, but not RAMP1 or RAMP3. Other regions within CRLR were not involved in trafficking interactions with RAMPs. These findings provide new insight into the trafficking interactions between accessory proteins such as RAMPs and their receptor partners.