RAMPs were first discovered based on their function in an expression cloning strategy, seeking a calcitonin gene-related peptide (CGRP) receptor (4
). It ultimately became clear that an accessory protein was necessary to be combined with the calcitonin receptor-like receptor (CLR) to facilitate its glycosylation and trafficking to the cell surface where it could bind and be activated by CGRP. This accessory protein was the first RAMP. This same type of survey was subsequently successful in identifying RAMP partners for the calcitonin receptor (CTR) and for the CLR, to provide molecular complexes that respond to amylin or adrenomedullin as well.
With both of these early recognized RAMP partners identified as family B GPCRs, many other members of the family have been screened for RAMP association. This was largely dependent on a fluorescent RAMP translocation assay in which receptor interaction with the RAMP, in transit through the biosynthetic compartments, leads to translocation of the RAMP to the plasma membrane. Indeed, this approach has been successful in demonstrating RAMP- and receptor-specific interactions where the PTH1, PTH2, VPAC1, and glucagon receptors all associate with one or more RAMPs, while the VPAC2, GHRH, GLP1 and GLP2 receptors do not appear to associate with any RAMPs (7
). More recently, association of RAMP1 and RAMP3 with the calcium-sensing receptor has been reported (8
). Despite this growing list of RAMP-receptor interactions, relatively little is known regarding the molecular mechanism(s) underlying the interaction. In the current study, we have identified the secretin receptor as a novel, specific partner for RAMP3 and have elucidated the interaction interface as TM6 and TM7 of the receptor.
To date, the best studied RAMP-GPCR interaction is that of CLR and RAMP1. For this receptor, a major component of the interaction interface is believed to be the amino-terminal domain of the receptor. Both the isolated amino-terminal domain of RAMP1 and the amino-terminal domain of RAMP1 linked to platelet-derived growth factor (PDGF) receptor transmembrane region are capable of eliciting cell surface expression of CLR and to engender a CGRP receptor phenotype (27
). This is further supported by loss of cell surface expression of CLR following mutagenesis of specific aromatic amino acids in helix 3 of the RAMP1 amino terminus (1
). A similar interaction between CLR and RAMP2 and RAMP3 is also likely as mutation of conserved amino-terminal histidine residues in these proteins also disrupts the ability of RAMPs and CLR to translocate to the cell surface, although the specific role of individual histidines can differ between RAMPs (28
). Nonetheless, the RAMP transmembrane region also played a contributory role in the interaction with CLR with both the isolated amino-terminal domain and the RAMP1-PDGF receptor chimera unable to fully reconstitute RAMP1 function (27
The critical role of the amino-terminal domains of CLR and the RAMPs contrasts with the lack of importance of these domains for interaction between the secretin receptor and RAMP3 observed in the current study. Deletion of either the secretin receptor amino terminus or the RAMP3 amino terminus had little effect on trafficking of RAMP3 to the cell surface. Use of isolated receptor transmembrane peptides and chimeras between the secretin receptor and the GLP1 receptor refined the key site of interaction to TM6 and TM7. A key role for the transmembrane region of RAMPs in receptor interaction has also been observed for the CTR where chimeras of RAMP1 and RAMP2 implicated the transmembrane region as the key determinant of the difference in strength of induction of amylin receptor phenotype (29
), however, the site of this interaction within the CTR has not been determined.
Phenotypically, all GPCR-RAMP interactions identified to date enable cell surface translocation of intracellularly retained RAMPs, which is not surprising as this is the underlying tenet for most assays for interaction. However, despite their initial description as modulators of peptide-binding specificity in association with CLR (4
), the alteration of binding profiles has only been observed for CLR and the closely-related CTR (1
). For other interacting receptors, including the VPAC1 and PTH1 receptor, no effect on peptide affinity has been observed (7
). A similar lack of effect by RAMP3 on secretin interaction with its receptor was found in the current study, with no apparent change in secretin potency across a broad range of assays including cAMP accumulation, intracellular calcium mobilization and phosphorylation of ERK1/2.
As with most receptors, the secretin receptor does not require RAMP co-expression for cell surface expression. Therefore, we cannot rule out the possibility that the lack of a specific demonstrable signaling phenotype in these assays is due to the expression of “uncomplexed” secretin receptor at the cell surface that overwhelms the response of RAMP3-secretin receptor complexes, since the percentage of complexed and uncomplexed receptor is unknown. However, under the varying conditions of assay for BRET and bimolecular fluorescence complementation, the level of interaction between RAMP3 and the secretin receptor was similar to that seen for CLR or CTR interaction with RAMP3, the classic RAMP-associating receptors. For the CTR, this level of interaction clearly enables resolution of signaling from uncomplexed and complexed receptor despite a high level of background uncomplexed CTR phenotype (6
Functional consequence of the secretin receptor-RAMP3 interaction was demonstrated in two additional assays. In the first series of studies, a trafficking-defective secretin receptor mutant was rescued by RAMP3 co-expression. This suggests that the RAMP can play a chaperone-like role for an interacting receptor, even when this is not required for routine cell surface receptor expression. In the second series of studies, secretin receptor competed for the RAMP3 interaction with CLR, an interaction that is critical for the establishment of a functional adrenomedullin receptor. Thus, we also need to be cognizant of how interacting molecules might saturate and compete for other functionally-important molecular associations.
The lack of effect in modulation of apparent secretin affinity is consistent with the lack of interaction between the amino-terminal domains of the secretin receptor and RAMP3. Indeed, it implies that lack of significant association between the amino-terminal domains may be the norm rather than the exception for most family B GPCR-RAMP interactions. The identification of TM6 and TM7 as the secretin-RAMP3 interface is therefore the first mechanistic guide to how most RAMP-family B receptor associations may occur.
Of note, the RAMP-secretin receptor interface is distinct from the homodimerization interface for secretin receptors that we recently identified as TM4 of this receptor (17
). Homodimerization of the secretin receptor appears to be important for efficient signaling of the receptor (17
), and our current data are consistent with an ability of RAMP3 to interact with an intact secretin receptor homodimer. For the CLR, elegant work has revealed that it is also a homodimer of this receptor that interacts with RAMPs, with the RAMP present as a monomer (32
), although the final stoichiometry of the complex is not clear. Assuming that there is conservation of function across family B GPCRs, these data provide support for the potential extrapolation of our current findings as a model for RAMP-family B GPCR transmembrane region interactions, however, an alternate interface involving TM1 and TM2 of CLR cannot be ruled out.
The other key RAMP-dependent phenotype that has been observed for CLR-RAMP3 interactions is an alteration to adrenomedullin-induced internalization and recycling of the AM2
). The AM2
receptor, but not the RAMP2-associated AM1
receptor, may be alternatively targeted for lysosomal degradation, rapidly recycled or not internalized at all depending upon interaction of the carboxyl-terminal RAMP3 PDZ domain with either NSF or NHERF1. Consequently, we studied internalization of the secretin receptor in the presence and absence of RAMPs and also in the context of overexpression of either NSF or NHERF1. However, internalization of the secretin receptor was not altered by RAMP3, either alone or in the presence of NSF or NHERF1, suggesting that the protein-protein interactions engendered by RAMP3 are contextual on the presentation of the PDZ epitope by its receptor partner.
The resolution of the site of transmembrane interaction between RAMP3 and the secretin receptor provides impetus towards understanding the molecular basis for the specificity of RAMP-GPCR interactions. If this interaction interface is confirmed for other GPCRs, it will enable a bioinformatic approach to prediction of RAMP-GPCR interactions. For example, the secretin receptor and the PTH2 receptor specifically interact with RAMP3, while the PTH1 and glucagon receptors specifically interact with RAMP2. There has been substantial work to explore the molecular basis of helix-helix interactions within the lipid bilayer (33
). Within the helical domain of the three RAMPs, there is approximately 30 percent identity of the amino acid residues and 60 percent homology. The differences between the RAMPs should be adequate to explain the specificity of interaction between a given GPCR and a given RAMP. Conservation of the site of transmembrane interaction would therefore allow rules to be developed for predicting such interactions.
Our data also provides further evidence for complexity in the functional consequence of RAMP-GPCR interaction with existing phenotypes varying greatly across interacting receptors and also many examples of lack of understanding of the functional significance of RAMP-GPCR interaction, as currently is the case for the secretin receptor-RAMP3 interaction. The broad distribution of RAMPs suggests that substantial work is still required to fully understand their role in physiology and disease.