The seven amino acids of human RAMP2 (86) and RAMP3 (59) are critical for agonist binding to human adrenomedullin receptors.

When co-expressed with a receptor activity-modifying protein (RAMP) accessory protein, calcitonin receptor-like receptor (CRLR) can function as a calcitonin gene-related peptide receptor (CRLR-RAMP1) or an adrenomedullin (AM) receptor (CRLR-RAMP2/3). Here we report on the structural domain(s) involved in selective AM binding that were examined using various RAMP chimeras and deletion mutants. Co-expression of chimeric RAMPs and CRLR in HEK293 cells revealed that residues 77-101, situated in the extracellular N-terminal domain of human RAMP2 (hRAMP2), were crucial for selective AM-evoked cAMP production. More detailed analysis showed that deletion of hRAMP2 residues 86-92 significantly attenuated high-affinity (125)I-AM binding and AM-evoked cAMP production despite full cell surface expression of the receptor heterodimer and that deletion of hRAMP3 residues 59-65 had a similar effect. There is little sequence identity between hRAMP3 residues 59-65 and hRAMP2 residues 86-92; moreover, substituting alanine for Trp(86) (Ala(87)), Met(88), Ile(89), Ser(90), Arg(91), or Pro(92) of hRAMP2 had no effect on AM-evoked cAMP production. It thus seems unlikely that any one amino acid residue is responsible for determining selective AM binding or that AM binds directly to these peptide segments. Instead these findings suggest that the respective seven-amino acid sequences confer selectivity either by directly contributing to the structure of ligand binding pocket or by allosteric modulation of the conformation of CRLR.     

N-Glycosylation and conserved cysteine residues in RAMP3 play a critical role for the functional expression of CRLR/RAMP3 adrenomedullin receptor

The calcitonin receptor-like receptor (CRLR) and receptor activity modifying protein-3 (RAMP3) can assemble into a CRLR/RAMP3 heterodimeric receptor that exhibits the characteristics of a high affinity adrenomedullin receptor. RAMP3 participates in adrenomedullin (AM) binding via its extracellular N-terminus characterized by the presence of six highly conserved cysteine residues and four N-glycosylation consensus sites. Here, we assessed the usage of these conserved residues in cotranslational modifications of RAMP3 and addressed their role in functional expression of the CRLR/RAMP3 receptor. Using a Xenopus oocyte expression system, we show that (i) RAMP3 is assembled with CRLR as a multiple N-glycosylated species in which two, three, or four consensus sites are used; (ii) elimination of all N-glycans in RAMP3 results in a significant inhibition of receptor [(125)I]AM binding and an increase in the EC(50) value for AM; (iii) several lines of indirect evidence indicate that each of the six cysteines is involved in disulfide bond formation; (iv) when all cysteines are mutated to serines, RAMP3 is N-glycosylated at all four consensus sites, suggesting that disulfide bond formation inhibits N-gylcosylation; and (v) elimination of all cysteines abolishes adrenomedullin binding and leads to a complete loss of receptor function. Our data demonstrate that cotranslational modifications of RAMP3 play a critical role in the function of the CRLR/RAMP3 adrenomedullin receptor.     

Structural basis for extracellular interactions between calcitonin receptor-like receptor and receptor activity-modifying protein 2 for adrenomedullin-specific binding

The calcitonin receptor-like receptor (CRLR), a class B GPCR, forms a heterodimer with receptor activity-modifying protein 2 (RAMP2), and serves as the adrenomedullin (AM) receptor to control neovascularization, while CRLR and RAMP1 form the calcitonin gene-related peptide (CGRP) receptor. Here, we report the crystal structures of the RAMP2 extracellular domain alone and in the complex with the CRLR extracellular domain. The CRLR-RAMP2 complex exhibits several intermolecular interactions that were not observed in the previously reported CRLR-RAMP1 complex, and thus the shape of the putative ligand-binding pocket of CRLR-RAMP2 is distinct from that of CRLR-RAMP1. The CRLR-RAMP2 interactions were confirmed for the full-length proteins on the cell surface by site-specific photo-crosslinking. Mutagenesis revealed that AM binding requires RAMP2 residues that are not conserved in RAMP1. Therefore, the differences in both the shapes and the key residues of the binding pocket are essential for the ligand specificity.     

Function of the cytoplasmic tail of human calcitonin receptor-like receptor in complex with receptor activity-modifying protein 2

Receptor activity-modifying protein 2 (RAMP2) enables calcitonin receptor-like receptor (CRLR) to form an adrenomedullin (AM)-specific receptor. Here we investigated the function of the cytoplasmic C-terminal tail (C-tail) of human (h)CRLR by co-transfecting its C-terminal mutants into HEK-293 cells stably expressing hRAMP2. Deleting the C-tail from CRLR disrupted AM-evoked cAMP production or receptor internalization, but did not affect [¹²⁵I]AM binding. We found that CRLR residues 428-439 are required for AM-evoked cAMP production, though deleting this region had little effect on receptor internalization. Moreover, pretreatment with pertussis toxin (100 ng/mL) led to significant increases in AM-induced cAMP production via wild-type CRLR/RAMP2 complexes. This effect was canceled by deleting CRLR residues 454-457, suggesting Gi couples to this region. Flow cytometric analysis revealed that CRLR truncation mutants lacking residues in the Ser/Thr-rich region extending from Ser⁴⁴⁹ to Ser⁴⁶⁷ were unable to undergo AM-induced receptor internalization and, in contrast to the effect on wild-type CRLR, overexpression of GPCR kinases-2, -3 and -4 failed to promote internalization of CRLR mutants lacking residues 449-467. Thus, the hCRLR C-tail is crucial for AM-evoked cAMP production and internalization of the CRLR/RAMP2, while the receptor internalization is dependent on the aforementioned GPCR kinases, but not Gs coupling.     

The myocardial response to adrenomedullin involves increased cAMP generation as well as augmented Akt phosphorylation

In this work we aimed to observe (1) the changes in adrenomedullin (AM) and its receptor system - calcitonin receptor-like receptor (CRLR) and receptor activity modifying proteins (RAMPs) - in myocardial ischemic injury and (2) the response of injuried myocardia to AM and the phosphorylation of Akt to illustrate the protective mechanism of AM in ischemic myocardia. Male SD rats were subcutaneously injected with isoproterenol (ISO) to induce myocardial ischemia. The mRNA levels of AM, CRLR, RAMP1, RAMP2 and RAMP3 were determined by RT-PCR. Protein levels of Akt, phosphor-Akt, CRLR, RAMP1, RAMP2 and RAMP3 were assayed by Western blot. Results showed that, compared with that of the controls, ISO-treated rats showed lower cardiac function and myocardial injury. The mRNA relative amount of AM, CRLR, RAMP1, RAMP2 and RAMP3 in the myocardia of ISO-treated rats was increased. The elevated mRNA levels of CRLR, RAMP1, RAMP2 and RAMP3 were positively correlated with AM content in injured myocardia. The protein levels of CRLR, RAMP1, RAMP2 and RAMP3 in injured myocardia were increased compared with that of control myocardia. AM-stimulated cAMP generation in myocardia was elevated in the ISO group, and was antagonized by AM(22-52) and CGRP(8-37). Western blot analyses revealed that AM significantly enhanced Akt phosphorylation in injured myocardia, which was blocked by pretreatment with AM(22-52) or CGRP(8-37). Ischemia-injured myocardia hyper-expressed AM and its receptors - CRLR, RAMP1, RAMP2 and RAMP3 - and the response of ischemic myocardia to AM was potentiated, and the level of Akt phosphorylation was also increased, which suggests that changes in cardiac AM/AM receptor might play an important role in the pathogenesis of myocardial ischemic injury.     

Protein-protein interaction and not glycosylation determines the binding selectivity of heterodimers between the calcitonin receptor-like receptor and the receptor activity-modifying proteins

The receptor activity-modifying proteins (RAMPs) and the calcitonin receptor-like receptor (CRLR) are both required to generate adrenomedullin (AM) and calcitonin gene-related peptide (CGRP) receptors. A mature, fully glycosylated, form of CRLR was associated with (125)I-CGRP binding, upon co-expression of RAMP1 and CRLR. In contrast, RAMP2 and -3 promoted the expression of smaller, core-glycosylated, CRLR forms, which were linked to AM receptor pharmacology. Since core glycosylation is classically a trademark of immature proteins, we tested the hypothesis that the core-glycosylated CRLR forms the AM receptor. Although significant amounts of core-glycosylated CRLR were produced upon co-expression with RAMP2 or -3, cross-linking experiments revealed that (125)I-AM only bound to the fully glycosylated forms. Similarly, (125)I-CGRP selectively recognized the mature CRLR species upon co-expression with RAMP1, indicating that the glycosylation does not determine ligand-binding selectivity. Our results also show that the three RAMPs lie close to the peptide binding pocket within the CRLR-RAMP heterodimers, since (125)I-AM and (125)I-CGRP were incorporated in RAMP2, -3, and -1, respectively. Cross-linking also stabilized the peptide-CRLR-RAMP ternary complexes, with the expected ligand selectivity, indicating that the fully processed heterodimers represent the functional receptors. Overall, the data indicate that direct protein-protein interactions dictate the pharmacological properties of the CRLR-RAMP complexes.     

Novel function for receptor activity-modifying proteins (RAMPs) in post-endocytic receptor trafficking

RAMPs (1-3) are single transmembrane accessory proteins crucial for plasma membrane expression, which also determine receptor phenotype of various G-protein-coupled receptors. For example, adrenomedullin receptors are comprised of RAMP2 or RAMP3 (AM1R and AM2R, respectively) and calcitonin receptor-like receptor (CRLR), while a CRLR heterodimer with RAMP1 yields a calcitonin gene-related peptide receptor. The major aim of this study was to determine the role of RAMPs in receptor trafficking. We hypothesized that a PDZ type I domain present in the C terminus of RAMP3, but not in RAMP1 or RAMP2, leads to protein-protein interactions that determine receptor trafficking. Employing adenylate cyclase assays, radioligand binding, and immunofluorescence microscopy, we observed that in HEK293 cells the CRLR-RAMP complex undergoes agonist-stimulated desensitization and internalization and fails to resensitize (i.e. degradation of the receptor complex). Co-expression of N-ethylmaleimide-sensitive factor (NSF) with the CRLR-RAMP3 complex, but not CRLR-RAMP1 or CRLR-RAMP2 complex, altered receptor trafficking to a recycling pathway. Mutational analysis of RAMP3, by deletion and point mutations, indicated that the PDZ motif of RAMP3 interacts with NSF to cause the change in trafficking. The role of RAMP3 and NSF in AM2R recycling was confirmed in rat mesangial cells, where RNA interference with RAMP3 and pharmacological inhibition of NSF both resulted in a lack of receptor resensitization/recycling after agonist-stimulated desensitization. These findings provide the first functional difference between the AM1R and AM2R at the level of post-endocytic receptor trafficking. These results indicate a novel function for RAMP3 in the post-endocytic sorting of the AM-R and suggest a broader regulatory role for RAMPs in receptor trafficking.     

Identification of the human receptor activity-modifying protein 1 domains responsible for agonist binding specificity

When co-expressed with receptor activity-modifying protein (RAMP) 1, calcitonin receptor-like receptor (CRLR) can function as a receptor for both calcitonin gene-related peptide (CGRP) and adrenomedullin (AM). To investigate the structural determinants of ligand binding specificity, we examined the extracellular domain of human (h) RAMP1 using various deletion mutants. Co-expression of the hRAMP1 mutants with hCRLR in HEK-293 cells revealed that deletion of residues 91-94, 96-100, or 101-103 blocked [125I]CGRP binding and completely abolished intracellular cAMP accumulation normally elicited by CGRP or AM. On the other hand, the deletion of residues 78-80 or 88-90 significantly attenuated only AM-evoked responses. In all of these cases, the receptor heterodimers were fully expressed at the cell surface. Substituting alanine for residues 91-103 one at a time had little effect on CGRP-induced responses, indicating that although this segment is essential for high affinity agonist binding to the receptors, none of the residues directly interacts with either CGRP or AM. This finding suggests that RAMPs probably determine ligand specificity by contributing to the structure of the ligand-binding pocket or by allosteric modulation of the conformation of the receptor. Interestingly, the L94A mutant up-regulated surface expression of the receptor heterodimer to a greater degree than wild-type hRAMP1, thereby increasing CGRP binding and signaling. L94A also significantly increased cell surface expression of the hRAMP1 deletion mutant D101-103 when co-transfected with hCRLR, and expression of a L94A/D101-103 double mutant markedly attenuated the activity of endogenous RAMP1 in HEK-293T cells.     

Functions of the extracellular histidine residues of receptor activity-modifying proteins vary within adrenomedullin receptors

Receptor activity-modifying protein (RAMP)-2 and -3 chaperone calcitonin receptor-like receptor (CRLR) to the plasma membrane, where together they form heterodimeric adrenomedullin (AM) receptors. We investigated the contributions made by His residues situated in the RAMP extracellular domain to AM receptor trafficking and receptor signaling by co-expressing hCRLR and V5-tagged-hRAMP2 or -3 mutants in which a His residue was substituted with Ala in HEK-293 cells. Flow cytometric analysis revealed that hRAMP2-H71A mediated normal hCRLR surface delivery, but the resultant heterodimers showed significantly diminished [¹²⁵I]AM binding and AM-evoked cAMP production. Expression of hRAMP2-H124A and -H127A impaired surface delivery of hCRLR, which impaired or abolishing AM binding and receptor signaling. Although hRAMP3-H97A mediated full surface delivery of hCRLR, the resultant heterodimers showed impaired AM binding and signaling. Other His residues appeared uninvolved in hCRLR-related functions. Thus, the His residues of hRAMP2 and -3 differentially govern AM receptor function.     

Functional relevance of G-protein-coupled-receptor-associated proteins,exemplified by receptor-activity-modifying proteins (RAMPs)

The calcitonin (CT) receptor (CTR) and the CTR-like receptor (CRLR) are close relatives within the type II family of G-protein-coupled receptors, demonstrating sequence identity of 50%. Unlike the interaction between CT and CTR, receptors for the related hormones and neuropeptides amylin, CT-gene-related peptide (CGRP) and adrenomedullin (AM) require one of three accessory receptor-activity-modifying proteins (RAMPs) for ligand recognition. An amylin/CGRP receptor is revealed when CTR is co-expressed with RAMP1. When complexed with RAMP3, CTR interacts with amylin alone. CRLR, initially classed as an orphan receptor, is a CGRP receptor when co-expressed with RAMP1. The same receptor is specific for AM in the presence of RAMP2. Together with human RAMP3, CRLR defines an AM receptor, and with mouse RAMP3 it is a low-affinity CGRP/AM receptor. CTR-RAMP1, antagonized preferentially by salmon CT-(8-32) and not by CGRP-(8-37), and CRLR-RAMP1, antagonized by CGRP-(8-37), are two CGRP receptor isotypes. Thus amylin and CGRP interact specifically with heterodimeric complexes between CTR and RAMP1 or RAMP3, and CGRP and AM interact with complexes between CRLR and RAMP1, RAMP2 or RAMP3.     

Adrenomedullin and its receptor complexes in remnant kidneys of rats with renal mass ablation: decreased expression of calcitonin receptor-like receptor and receptor-activity modifying protein-3.

Adrenomedullin (AM) has vasodilator and diuretic actions, similarly to natriuretic peptides. AM receptor complexes are composed of calcitonin receptor-like receptor (CRLR) and receptor-activity modifying protein-2 (RAMP2), or CRLR and RAMP3. We aimed to know whether gene expression of AM and AM receptor complexes are regulated in kidneys under pathophysiological conditions. Expression of AM, RAMP2, RAMP3 and CRLR mRNA was studied in the remnant kidney of rats with renal mass ablation using competitive quantitative RT-PCR techniques. Partial cloning was performed to determine the rat RAMP3 nucleotide sequence. In normal rat kidneys, expression levels of RAMP2, RAMP3, CRLR and AM mRNAs were 26.5 +/- 1.9 mmol/mole of GAPDH, 7.7 +/- 0.9 mmol/mole of GAPDH, 3.6 +/- 0.2 mmol/mole of GAPDH and 0.57 +/- 0.03 mmol/mole of GAPDH (mean +/- SE, n = 6), respectively. RAMP3 mRNA levels decreased significantly to about 50% and about 70% of control (sham-operated rats) 4 days and 14 days after 5/6 nephrectomy, respectively. CRLR mRNA levels also decreased significantly to about 30% and about 43% of control. Sodium intake restriction had no significant effects on the RAMP3 and CRLR gene expression. On the other hand, RAMP2 mRNA expression in the kidney was suppressed by sodium intake restriction regardless of nephrectomy, while RAMP2 levels in the remnant kidney were not significantly changed by 5/6 nephrectomy. Neither 5/6 nephrectomy or sodium intake restriction had any significant effects on the AM gene expression in the kidney. The present study showed that expression of mRNAs encoding AM, RAMP2, RAMP3 and CRLR were differentially regulated in remnant kidneys of rats with renal mass ablation.     

Functions of the cytoplasmic tails of the human receptor activity-modifying protein components of calcitonin gene-related peptide and adrenomedullin receptors

Receptor activity-modifying proteins (RAMPs) enable calcitonin receptor-like receptor (CRLR) to function as a calcitonin gene-related peptide receptor (CRLR/RAMP1) or an adrenomedullin (AM) receptor (CRLR/RAMP2 or -3). Here we investigated the functions of the cytoplasmic C-terminal tails (C-tails) of human RAMP1, -2, and -3 (hRAMP1, -2, and -3) by cotransfecting their C-terminal deletion or progressive truncation mutants into HEK-293 cells stably expressing hCRLR. Deletion of the C-tail from hRAMP1 had little effect on the surface expression, function, or intracellular trafficking of the mutant heterodimers. By contrast, deletion of the C-tail from hRAMP2 disrupted transport of hCRLR to the cell surface, resulting in significant reductions in (125)I-hAM binding and evoked cAMP accumulation. The transfection efficiency for the hRAMP2 mutant was comparable with that for wild-type hRAMP2; moreover, immunocytochemical analysis showed that the mutant hRAMP2 remained within the endoplasmic reticulum. FACS analysis revealed that deleting the C-tail from hRAMP3 markedly enhances AM-evoked internalization of the mutant heterodimers, although there was no change in agonist affinity. Truncating the C-tails by removing the six C-terminal amino acids of hRAMP2 and -3 or exchanging their C-tails with one another had no effect on surface expression, agonist affinity, or internalization of hCRLR, which suggests that the highly conserved Ser-Lys sequence within hRAMP C-tails is involved in cellular trafficking of the two AM receptors. Notably, deleting the respective C-tails from hRAMPs had no effect on lysosomal sorting of hCRLR. Thus, the respective C-tails of hRAMP2 and -3 differentially affect hCRLR surface delivery and internalization.     

Visualization of the calcitonin receptor-like receptor and its receptor activity-modifying proteins during internalization and recycling

Expression of the calcitonin receptor-like receptor (CRLR) and its receptor activity modifying proteins (RAMPs) can produce calcitonin gene-related peptide (CGRP) receptors (CRLR/RAMP1) and adrenomedullin (AM) receptors (CRLR/RAMP2 or -3). A chimera of the CRLR and green fluorescent protein (CRLR-GFP) was used to study receptor localization and trafficking in stably transduced HEK 293 cells, with or without co-transfection of RAMPs. CRLR-GFP failed to generate responses to CGRP or AM without RAMPs. Furthermore, CRLR-GFP was not found in the plasma membrane and its localization was unchanged after agonist exposure. When stably coexpressed with RAMPs, CRLR-GFP appeared on the cell surface and was fully active in intracellular cAMP production and calcium mobilization. Agonist-mediated internalization of CRLR-GFP was observed in RAMP1/CGRP or AM, RAMP2/AM, and RAMP3/AM, which occurred with similar kinetics, indicating the existence of ligand-specific regulation of CRLR internalization by RAMPs. This internalization was strongly inhibited by hypertonic medium (0.45 m sucrose) and paralleled localization of rhodamine-labeled transferrin, suggesting that CRLR endocytosis occurred predominantly through a clathrin-dependent pathway. A significant proportion of CRLR was targeted to lysosomes upon binding of the ligands, and recycling of the internalized CRLR was not efficient. In HEK 293 cells stably expressing CRLR-GFP and Myc-RAMPs, these rhodamine-labeled RAMPs were co-localized with CRLR-GFP in the presence and absence of the ligands. Thus, the CRLR is endocytosed together with RAMPs via clathrin-coated vesicles, and both the internalized molecules are targeted to the degradative pathway.     

Interaction of calcitonin-gene-related peptide with its receptors

The receptor for calcitonin-gene-related peptide (CGRP) is a heterodimer formed by calcitonin-receptor-like receptor (CRLR), a type II (family B) G-protein-coupled receptor, and receptor-activity-modifying protein 1 (RAMP1), a single-membrane-pass protein. It is likely that the first seven or so amino acids of CGRP (which form a disulphide-bonded loop) interact with the transmembrane domain of CRLR to cause receptor activation. The rest of the CGRP molecule falls into three domains. Residues 28-37 and 8-18 are normally required for high-affinity binding, while residues 19-27 form a hinge region. The 28-37 region is almost certainly in direct contact with the receptor; 8-18 may make additional receptor contacts or may stabilize an appropriate conformation of 28-37. It is likely that these regions of CGRP interact both with CRLR and with the extracellular domain of RAMP1.     

Differential gene expression of adrenomedullin receptors in pressure- and volume-overloaded heart--role of angiotensin II

Left ventricular (LV) adrenomedullin (AM) gene expression differs between pressure overload (POL) and volume overload (VOL) and angiotensin II could be a critical stimulator of AM gene expression in POL and VOL models. Calcitonin receptor-like receptor (CRLR) co-expressed with receptor activity modifying protein 2 (RAMP2) or RAMP3 functions as an AM receptor. Levels of CRLR, RAMP2 and RAMP3 mRNA that were significantly increased within 24 h returned to the basal level at 5 days after the imposition of POL in the present study. In contrast, mRNA levels of CRLR and RAMP2 gradually increased over 6 weeks after the imposition of VOL. Continuous infusion of angiotensin II stimulated LV AM gene and AM receptor gene expression independently of LV peak-systolic and LV end-diastolic pressure. The gene expression of LV AM receptors increased in different types of cardiac overload. The present study revealed an intimate association between the AM signaling system and angiotensin II.     

Receptor activity-modifying protein (RAMP) isoform-specific regulation of adrenomedullin receptor trafficking by NHERF-1

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.     

Glycosylation of human CRLR at Asn123 is required for ligand binding and signaling

Calcitonin receptor-like receptor (CRLR) constitutes either a CGRP receptor when complexed with receptor activity-modifying protein 1 (RAMP1) or an adrenomedullin receptor when complexed with RAMP2 or RAMP3. RAMP proteins modify the glycosylation status of CRLR and determine their receptor specificity; when treated with tunicamycin, a glycosylation inhibitor, CHO-K1 cells constitutively expressing both RAMP2 and CRLR lost the capacity to bind adrenomedullin. Similarly, in HEK293 EBNA cells constitutively expressing RAMP1/CRLR receptor complex CGRP binding was remarkably inhibited. Whichever RAMP protein was co-expressing with CRLR, the ligand binding was sensitive to tunicamycin. There are three putative Asn-linked glycosylation sites in the extracellular, amino terminal domain of CRLR at positions 66, 118 and 123. Analysis of CRLR mutants in which Gln was substituted for selected Asn residues showed that glycosylation of Asn123 is required for both the binding of adrenomedullin and the transduction of its signal. Substituting Asn66 or Asn118 had no effect. FACS analysis of cells expressing FLAG-tagged CRLRs showed that disrupting Asn-linked glycosylation severely affected the transport of the CRLR protein to the cell surface on N66/118/123Q mutant, and slightly reduced the level of the cell surface expression of N123Q mutant compared with wild-type CRLR. But other single mutants (N66Q, N118Q) had no effect for other single mutants. Our data shows that glycosylation of Asn66 and Asn118 is not essential for ligand binding, signal transduction and cell surface expression, and Asn123 is important for ligand binding and signal transduction rather than cell surface expression. It thus appears that glycosylation of Asn123 is required for CRLR to assume the appropriate conformation on the cell surface through its interaction with RAMPs.     

Expression of adrenomedullin in human epicardial adipose tissue: role of coronary status

Epicardial white adipose tissue (eWAT) is in close contact with coronary vessels and therefore could alter coronary homeostasis. Adrenomedullin (AM) is a potent vasodilatator and antioxidative peptide which has been shown to play a cytoprotective role in experimental models of acute myocardial infarction. We studied, using immunohistochemistry and qRT-PCR, the expression of AM and its receptors calcitonin receptor-like receptor (CRLR), and receptor activity-modifying protein (RAMP)2 and -3 in paired biopsies of subcutaneous WAT (sWAT) and eWAT obtained from patients with coronary artery disease (CAD) or without CAD (NCAD). In eWAT obtained from NCAD or CAD patients, immunoreactivity for AM, CRLR, and RAMP2 and -3 was detected in blood vessel walls and isolated stromal cells close to adipocytes. Some of the AM positive stromal cells colocalized CD68 immunoreactivity. eWAT from CAD patients showed increased AM immunoreactivity and AM gene expression. CRLR mRNA levels were comparable in sWAT of both groups and decreased by 40-50% in eWAT, irrespectively of the coronary status. RAMP2 mRNA concentrations did not change while RAMP3 mRNA levels increased in sWAT from CAD patients. There was a positive linear relationship between eWAT 11beta-hydroxysteroid dehydrogenase type 1 mRNA (11beta-HSD-1, the enzyme that converts inactive to active glucocorticoids) and AM mRNA. In conclusion, we demonstrate that AM and its receptors are expressed in eWAT. Our data suggest that eWAT AM, which could originate from macrophages, is related to 11beta-HSD-1 expression. AM synthesis, which is increased in eWAT during chronic CAD in humans, can play a cardioprotective role.