Agonist-promoted Internalization of a Ternary Complex between Calcitonin Receptor-like Receptor, Receptor Activity-modifying Protein 1 (RAMP1), and β-Arrestin

Abstract

The calcitonin receptor-like receptor (CRLR) is a seven-transmembrane domain (7TM) protein that requires the receptor activity-modifying protein 1 (RAMP1) to be expressed at the cell surface as a functional calcitonin gene-related peptide (CGRP) receptor. Although dimerization between the two molecules is well established, very little is known concerning the trafficking of this heterodimer upon receptor activation. Also, the subcellular localization and biochemical state of this ubiquitously expressed protein, in the absence of CRLR, remains poorly characterized. Here we report that when expressed alone RAMP1 is retained inside the cells where it is found in the endoplasmic reticulum and the Golgi predominantly as a disulfide-linked homodimer. In contrast, when expressed with CRLR, it is targeted to the cell surface as a 1:1 heterodimer with the 7TM protein. Although heterodimer formation does not involve intermolecular disulfide bonds, RAMP-CRLR association promotes the formation of intramolecular disulfide bonds within RAMP1. CGRP binding and receptor activation lead to the phosphorylation of CRLR and the internalization of the receptor as a stable complex. The internalization was found to be both dynamin-and ␤-arrestin-dependent, indicating that the formation of a ternary complex between CRLR, RAMP1, and ␤-arrestin leads to clathrin-coated pit-mediated endocytosis. These results therefore indicate that although atypical by its heterodimeric composition and its targeting to the plasma membrane, the CGRP receptor shares endocytotic mechanisms that are common to most classical 7TM receptors.The calcitonin gene-related peptide (CGRP) 1 is a member of the calcitonin family of regulatory peptides (1-3). Although a cDNA encoding the calcitonin receptor was cloned over 10 years ago (4), it was only recently that McLatchie et al. (5) established that co-expression of an orphan calcitonin-receptor like receptor (CRLR) with a newly discovered protein named receptor activity-modifying protein 1 (RAMP1) created a CGRP receptor. CRLR shares 55% amino acid sequence identity with the calcitonin receptor. Both belong to the family B of seventransmembrane domain (7TM) receptor. The family also includes the receptors for parathyroid hormone, parathyroid hormone-related protein, secretin, glucagon, and vasoactive intestinal polypeptide/pituitary adenylate cyclase activating polypeptide (6, 7). RAMP1, for its part, is a member of a family that comprises 3 members designed RAMP1, RAMP2, and RAMP3. These small intrinsic membrane proteins share less than 30% of sequence identity and possess a large extracellular N terminus (ϳ100 amino acids), a single transmembrane domain, and a very short intracellular domain (10 amino acids) (5). One of the roles proposed for these proteins is that of chaperone/escort promoting the cell surface targeting of CRLR (5, 8). Interestingly, like CRLR, RAMPs are also retained intracellularly when expressed alone suggesting that an interaction between the two proteins is essential to their common cell...

Negative Regulation of TLR Responses by the Neuropeptide CGRP Is Mediated by the Transcriptional Repressor ICER

“…First-strand cDNA was synthesized from 1 g of total RNA using a mixture of oligo(dT) [12][13][14][15][16][17][18] and random hexamer primers and Superscript Reverse Transcriptase (Invitrogen Life Technologies). For amplification, the qPCR Mastermix Plus for SYBR Green I without UNG (Eurogentec) was used to detect accumulation of PCR products during cycling on an Applied Biosystems 7300 cycler.…”

“…Alternatively, the CGRP receptor may activate phospholipase C-␤1 via G␣ q/11 proteins, causing calcium mobilization (13). Following prolonged CGRP exposure, internalization of a ternary complex between CRLR-RAMP1 and ␤-arrestin through clathrincoated pit-mediated endocytosis was observed (14).…”

Accessory proteins are vital for the functional expression of certain G protein-coupled receptors

“…However neither RAMP2 nor RAMP3 is known to change the glycosylation pattern to a significant loss in CGRP potency despite normal trafficking of the receptor complex to the plasma membrane (Hilairet et al, 2001). Studies have also shown that the CLR, RAMP1 and arrestins can exist in a ternary complex in HEK 293 cells (Hilairet et al, 2001).…”

“…Studies have also shown that the CLR, RAMP1 and arrestins can exist in a ternary complex in HEK 293 cells (Hilairet et al, 2001). This has led to the suggestion that RAMPs can associate with the CLR throughout its entire life cycle being an essential component of the desensitisation and internalisation machinary.…”

“…Studies have shown that RAMP1 is involved in the terminal glycosylation of the CLR and that RAMP1 can directly interact with both the immature and mature receptors with a stable interaction at the cell surface with the mature CLR (Hilairet et al, 2001). …”