The receptor activity-modifying protein (RAMP) family was first reported in 1998 during attempts to identify the cell surface receptor for a neuropeptide known as calcitonin gene-related peptide (CGRP) (McLatchie et al. 1998). Formerly, a protein known as the calcitonin receptor-like receptor (CLR) was thought to be the receptor for CGRP, but no study had convincingly shown that this was the case. McLatchie and colleagues were able to show that CLR needs RAMP1 for a CGRP receptor to be formed. RAMP1 assists CLR in reaching the cell surface. Thus, RAMP1 and CLR together at the cell surface form the receptor for CGRP, which binds and activates this protein complex, leading to downstream signaling events such as an accumulation of intracellular cAMP. In this same study, two other related proteins were found, named RAMP2 and RAMP3. Each of these proteins could also assist CLR in reaching the cell surface, but remarkably, CGRP was less effective at activating these protein complexes. Instead, a peptide similar to CGRP, called adrenomedullin (AM), preferentially activated them. Thus, RAMPs can be considered as “pharmacological switches” by virtue of their ability to change the peptide hormone for which CLR has a preference.
Pharmacological Importance of RAMPs
At first it was thought that the way RAMPs could change peptide preference was by changing the conformation of the receptor protein, CLR, but it is now known that RAMPs also play their own role in binding CGRP and AM and also small molecule drugs (Sexton et al. 2009; Hay and Pioszak 2016). For instance, RAMP1 and CLR both participate in the binding of the drugs Olcegepant and Telcagepant. This makes the drugs relatively selective for the CGRP receptor; they have lower affinity for the two AM receptors or for the RAMP1-based AMY1 receptor. RAMPs also change the pharmacology of another receptor, the calcitonin receptor (CTR). This is the closest protein relative to CLR and can also interact with all three RAMPs. In this case, CTR is a receptor for the peptide hormone, calcitonin, when it is expressed alone in cells. However, when CTR is expressed with RAMPs, the resulting RAMP/CTR complexes have a preference for a peptide hormone known as amylin (Fig. 1) (Poyner et al. 2002). The RAMP1/CTR complex also has very high affinity for CGRP (Walker et al. 2015).
RAMPs and Cellular Signaling
CLR and CTR are proteins known as G protein-coupled receptors (GPCRs). As their name suggests, these proteins interact with G proteins (guanine nucleotide binding proteins) to initiate intracellular signaling. There are several types of G proteins that initiate different downstream signaling events, and many receptors can interact with more than one G protein to regulate cellular function. For instance, Gs G proteins activate adenylate cyclase to cause increases in cAMP, while Gq G proteins activate phospholipase C to hydrolyze membrane inositol phosphates to form inositol trisphosphate ( IP3 Receptors) and diacyl glycerol (DAG); these in turn mobilize intracellular calcium and activate various kinase proteins. Both CLR and CTR can couple to Gs and signal via adenylate cyclase/cAMP pathway. In the case of the RAMP/CTR complexes, that is, the amylin receptors, the amount of amylin binding can be influenced by G protein type in a RAMP-dependent manner (Morfis et al. 2008), and conversely RAMP interaction with the CTR can change the receptor preference for different G proteins. For example, the RAMP1/CTR (AMY1) and RAMP3/CTR (AMY3) receptors have greater relative preference for Gs over Gq G proteins than the CTR when expressed alone (Morfis et al. 2008), and this may change to overall cellular response to receptor activation.
The activity of a GPCR must be carefully controlled to make sure that cells are kept responsive when they need to be. In many cases, a process called desensitization occurs after a GPCR has been activated, which reduces the amount of signal that the receptor can generate. Following this, GPCRs are often removed from the cell surface membrane; the receptors are internalized. The GPCR may then be returned to the cell surface and, thus, recycled or it can be degraded and not returned. RAMPs can also modulate these internalization and recycling processes. RAMP3 has a specific sequence of amino acids, a PDF-like domain, in its intracellular domain. This allows it to interact with different regulatory proteins to RAMP1 and RAMP2, such as N-ethylmaleimide-sensitive factor (Bomberger et al. 2005). Therefore, in model systems, RAMP3/CLR (AM2 receptor) complexes can be recycled unlike AM1 and CGRP receptors.
RAMP Interactions with Other Receptors
There is accumulating research which shows that RAMPs may have much broader roles and control aspects of the activity of many receptors (Hay and Pioszak 2016). For example, the calcium sensing receptor that is unrelated to CLR or CTR requires RAMP1 or RAMP3 for it to reach the cell surface and therefore signal (Bouschet et al. 2005). The VPAC1 receptor can associate with RAMP2 and this leads to enhanced, agonist-mediated phosphoinositide breakdown (Christopoulos et al. 2003).
The Structure of RAMPs
RAMPs appear to be widely expressed in mammals, although there are actually very few studies where the proteins themselves have been measured, rather than mRNA. This has been because there have been few reliable antibodies. This has meant that it has been difficult to properly colocalize RAMPs and receptors. Studies have reported the co-expression of RAMP1 and CLR or RAMP1 and CTR in neurons of the human and rodent trigeminal ganglion, which is involved in pain transmission (Eftekhari et al. 2010; Walker et al. 2015). More studies of this nature are needed to confirm the physiological significance of the AM and amylin receptor subtypes and the interactions of RAMPs with other receptors.
Several animal models of RAMP under or overexpression have been generated. Mice that selectively overexpress human RAMP1 in neurons are sensitized to CGRP, whereas those that overexpress RAMP2 in the smooth muscle were sensitized to the effects of AM (Tam et al. 2006; Zhang et al. 2007). These types of observation help to confirm that these are valid components of AM and CGRP receptors in vivo. Mice that genetically lack RAMP2 (RAMP2 knockout mice) have severe defects and show that RAMP2 is essential for the blood and lymphatic vascular systems to develop properly in the embryo (Fritz-Six et al. 2008). Interestingly, RAMP3 knockout mice do not have any obvious phenotype, suggesting that RAMP3, and the AM2 receptors which it form with CLR, has different functions to the AM1 receptor (Dackor et al. 2007).
There is some evidence that a form of CGRP first evolved in insects. In Drosophila melanogaster, the protein CG17415 shows homology to CLR. It is activated by the diuretic hormone DH31 and this response can be amplified when it is co-expressed with human RAMP1 or RAMP2 (Johnson et al. 2005). However, no ortholog of a RAMP has yet been identified in Drosophila. RAMPs are certainly present in bony fish, where they are found with homologues of CLR and AM. The functions of RAMPs have been best studied in the pufferfish, Takifugu obscurus. This expresses five forms of AM, three forms of CLR, and five RAMPs. The expanded AM/RAMP/CLR family appears to be involved in fluid homeostasis (Nag et al. 2006). The evolutionary history of RAMPs between insects and fish remains obscure.
This entry has provided a snapshot of what RAMPs are and what they are currently understood to do. Most of this research has been performed in isolated cellular systems because these have been the models that have been available. It is important to consider whole organism studies in more detail so it can be fully appreciated how broad the functions of these proteins may be in physiology and disease.
- Booe JM, Walker CS, Barwell J, Kuteyi G, Simms J, Jamaluddin MA, Warner ML, Bill RM, Harris PW, Brimble MA, Poyner DR, Hay DL, Pioszak AA. Structural basis for receptor activity-modifying protein-dependent selective peptide recognition by a G protein-coupled receptor. Mol Cell. 2015;58(6):1040–52. doi: 10.1016/j.molcel.2015.04.018.PubMedPubMedCentralCrossRefGoogle Scholar
- Eftekhari S, Salvatore CA, Calamari A, Kane SA, Tajti J, Edvinsson L. Differential distribution of calcitonin gene-related peptide and calcitonin gene-related peptide receptor components (calcitonin receptor-like receptor and receptor activity-modifying protein 1) in the human trigeminal ganglion. Neuroscience. 2010;169:683–96.PubMedCrossRefGoogle Scholar
- Koth CM, Abdul-Manan N, Lepre CA, Connolly PJ, Yoo S, Mohanty AK, Lippke JA, Zwahlen J, Coll JT, Doran JD, Garcia-Guzman M, Moore JM. Refolding and characterization of a soluble ectodomain complex of the calcitonin gene-related peptide receptor. Biochemistry. 2010;49:1862–72.PubMedCrossRefGoogle Scholar
- Nag K, Kato A, Nakada T, Hoshijima K, Mistry AC, Takei Y, Hirose S. Molecular and functional characterization of adrenomedullin receptors in pufferfish. Am J Phys Regul Integr Comp Phys. 2006;290:R467–78.Google Scholar
- Tam CW, Husmann K, Clark NC, Clark JE, Lazar Z, Ittner LM, Götz J, Douglas G, Grant AD, Sugden D, Poston L, Poston R, McFadzean I, Marber MS, Fischer JA, Born W, Brain SD. Enhanced vascular responses to adrenomedullin in mice overexpressing receptor-activity-modifying protein 2. Circ Res. 2006;98:262–70.PubMedCrossRefGoogle Scholar