Abstract
Receptor activity-modifying proteins (RAMPs 1-3) are single transmembrane accessory proteins critical to various G-protein coupled receptors for plasma membrane expression and receptor phenotype. A functional receptor for the vasodilatory ligand, adrenomedullin (AM), is comprised of RAMP2 or RAMP3 and calcitonin receptor-like receptor (CRLR). It is now known that RAMP3 protein-protein interactions regulate the recycling of the AM2 receptor. The major aim of this study was to identify other interaction partners of RAMP3 and determine their role in CRLR-RAMP3 trafficking. Trafficking of G-protein-coupled receptors has been shown to be regulated by the Na+/H+ exchanger regulatory factor-1 (NHERF-1), an adaptor protein containing two tandem PSD-95/Discs-large/ZO-1 homology (PDZ) domains. In HEK 293T cells expressing the AM2 receptor, the complex undergoes agonist-induced desensitization and internalization. However, in the presence of NHERF-1, although the AM receptor (CRLR/RAMP3) undergoes desensitization, the internalization of the receptor complex is blocked. Overlay assays and mutational analysis indicated that RAMP3 and NHERF-1 interact via a PDZ type I domain on NHERF-1. The internalization of the CRLR-RAMP complex was not affected by NHERF-1 when CRLR was co-expressed with RAMP1 or RAMP2. Mutation of the ezrin/radixin/moesin (ERM) domain on NHERF-1 indicated that NHERF-1 inhibits CRLR/RAMP3 complex internalization by tethering the complex to the actin cytoskeleton. When examined in a primary culture of human proximal tubule cells endogenously expressing the CRLR-RAMP3 complex and NHERF-1, the CRLR-RAMP complex desensitizes but is unable to internalize upon agonist stimulation. Knock-down of either RAMP3 or NHERF-1 by RNA interference technology enabled agonist-induced internalization of the CRLR-RAMP complex. These results, using both endogenous and overexpressed cellular models, indicate a novel function for NHERF-1 and RAMP3 in the internalization of the AM receptor and suggest additional regulatory mechanisms for receptor trafficking.
Highlights
Introduction□S The on-line version of this article (available at http://www.jbc.org) contains a supplemental table showing receptor complex expression levels with transient transfection in HEK 293T cells
□S The on-line version of this article contains a supplemental table showing receptor complex expression levels with transient transfection in HEK 293T cells
We have examined the role of another protein, namely Na؉/H؉ exchanger regulatory factor-1 (NHERF-1), on agonist-induced trafficking of the calcitonin receptor-like receptor (CRLR)/RAMP3 complex
Summary
□S The on-line version of this article (available at http://www.jbc.org) contains a supplemental table showing receptor complex expression levels with transient transfection in HEK 293T cells. The identification of RAMP interactions with additional members of the Class II GPCR family and RAMP expression in cell lines lacking CRLR have raised the possibility of novel functions for RAMPs in GPCR regulation. It has been shown in other GPCR systems that interactions with PSD-95/Discs-large/ZO-1 homology (PDZ) domain proteins are responsible for altering the receptor trafficking after agonist stimulation [7,8,9]. We show here that overexpression of NHERF-1 in HEK 293T cells alters the trafficking pattern of the receptor complex to block the internalization of the receptor by tethering the receptor complex to the actin cytoskeleton via interactions between RAMP3 and NHERF-1 through a type I PDZ domain. Knocking down NHERF-1 or RAMP3 expression with RNA interference causes the receptor to undergo internalization upon agonist treatment, suggesting critical roles for both NHERF-1 and RAMP3 in receptor internalization
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