Adrenomedullin: Continuing to explore cardioprotection.

Adrenomedullin (AM), a peptide isolated from an extract of human pheochromocytoma, comprises 52 amino acids with an intramolecular disulfide bond and amidation at the carboxy-terminus. AM is present in various tissues and organs in rodents and humans, including the heart. The peptide concentration increases with cardiac hypertrophy, acute myocardial infarction, and overt heart failure in the plasma and the myocardium. The principal function of AM in the cardiovascular system is the regulation of the vascular tone by vasodilation and natriuresis via cyclic adenosine monophosphate-dependent or -independent mechanism. In addition, AM may possess unique properties that inhibit aldosterone secretion, oxidative stress, apoptosis, and stimulation of angiogenesis, resulting in the protection of the structure and function of the heart. The AM receptor comprises a complex between calcitonin receptor-like receptor (CLR) and receptor activity-modifying protein (RAMP) 2 or 3, and the AM-CLR/RAMP2 system is essential for heart development during embryogenesis. Small-scale clinical trials have proven the efficacy and safety of recombinant AM peptide therapy for heart failure. Gene delivery and a modified AM peptide that prolongs the half-life of the native peptide could be an innovative method to improve the efficacy and benefit of AM in clinical settings. In this review, we focus on the pathophysiological roles of AM and its receptor system in the heart and describe the advances in AM and proAM-derived peptides as diagnostic biomarkers as well as the therapeutic application of AM and modified AM for cardioprotection.

Calcitonin-typical suppression of osteoclastic activity by amphioxus calcitonin superfamily peptides and insights into the evolutionary conservation and diversity of their structures.

Calcitonin (CT) is a hormone that decreases serum calcium level by suppressing osteoclastic activity in the vertebrate bone. In vertebrates, the structure-function relationship of CTs has been studied extensively. We recently identified three CT superfamily peptides, Bf-CTFP1 to 3, and clarified the molecular and functional characteristics of their receptor and receptor activity-modifying protein in amphioxus, Branchiostoma floridae. However, the CT activity of Bf-CTFPs has yet to be investigated. In the present study, a functional analysis of Bf-CTFPs was performed using goldfish scales having both osteoclasts and osteoblasts. All Bf-CTFPs suppressed osteoclastic activity via a goldfish CT receptor. Although the primary amino acid sequences of the Bf-CTFPs showed low sequence similarity to vertebrate CTs, Bf-CTFP1 to 3 share three amino acids, Thr25, Thr27, and Pro32-NH2, that are required for receptor binding, with salmon CT. Moreover, homology model analysis revealed that the Bf-CTFPs form alpha-helical structures. The alpha-helical position and length of Bf-CTFP1 and 2 were conserved with those of a highly potent ligand, teleost CT. Interestingly, the composition of the alpha-helix of Bf-CTFP3 differed from those of teleost CT, despite that the action of Bf-CTFP3 on goldfish scales was the same as that of Bf-CTFP1 and 2. Collectively, the present study provides new insights into the structure-function relationship of CT and its functional evolution in chordates.

Evidence for Conservation of the Calcitonin Superfamily and Activity-regulating Mechanisms in the Basal Chordate Branchiostoma floridae: INSIGHTS INTO THE MOLECULAR AND FUNCTIONAL EVOLUTION IN CHORDATES.

The calcitonin (CT)/CT gene-related peptide (CGRP) family is conserved in vertebrates. The activities of this peptide family are regulated by a combination of two receptors, namely the calcitonin receptor (CTR) and the CTR-like receptor (CLR), and three receptor activity-modifying proteins (RAMPs). Furthermore, RAMPs act as escort proteins by translocating CLR to the cell membrane. Recently, CT/CGRP family peptides have been identified or inferred in several invertebrates. However, the molecular characteristics and relevant functions of the CTR/CLR and RAMPs in invertebrates remain unclear. In this study, we identified three CT/CGRP family peptides (Bf-CTFPs), one CTR/CLR-like receptor (Bf-CTFP-R), and three RAMP-like proteins (Bf-RAMP-LPs) in the basal chordate amphioxus (Branchiostoma floridae). The Bf-CTFPs were shown to possess an N-terminal circular region typical of the CT/CGRP family and a C-terminal Pro-NH2. The Bf-CTFP genes were expressed in the central nervous system and in endocrine cells of the midgut, indicating that Bf-CTFPs serve as brain and/or gut peptides. Cell surface expression of the Bf-CTFP-R was enhanced by co-expression with each Bf-RAMP-LP. Furthermore, Bf-CTFPs activated Bf-CTFP-R·Bf-RAMP-LP complexes, resulting in cAMP accumulation. These results confirmed that Bf-RAMP-LPs, like vertebrate RAMPs, are prerequisites for the function and translocation of the Bf-CTFP-R. The relative potencies of the three peptides at each receptor were similar. Bf-CTFP2 was a potent ligand at all receptors in cAMP assays. Bf-RAMP-LP effects on ligand potency order were distinct to vertebrate CGRP/adrenomedullin/amylin receptors. To the best of our knowledge, this is the first molecular and functional characterization of an authentic invertebrate CT/CGRP family receptor and RAMPs.

Biological properties of adrenomedullin conjugated with polyethylene glycol.

Adrenomedullin (AM) is a vasodilator peptide with pleiotropic effects, including cardiovascular protection and anti-inflammation. Because of these beneficial effects, AM appears to be a promising therapeutic tool for human diseases, while intravenous injection of AM stimulates sympathetic nerve activity due to short-acting potent vasodilation, resulting in increased heart rate and renin secretion. To lessen these acute reactions, we conjugated the N-terminal of human AM peptide with polyethylene glycol (PEG), and examined the biological properties of PEGylated AM in the present study. PEGylated AM stimulated cAMP production, an intracellular second messenger of AM, in cultured human embryonic kidney cells expressing a specific AM receptor in a dose-dependent manner, as did native human AM. The pEC50 value of PEGylated AM was lower than human AM, but no difference was noted in maximum response (Emax) between the PEGylated and native peptides. Intravenous bolus injection of 10nmol/kg PEGylated AM lowered blood pressure in anesthetized rats, but the acute reduction became significantly smaller by PEGylation as compared with native AM. Plasma half-life of PEGylated AM was significantly longer than native AM both in the first and second phases in rats. In summary, N-terminal PEGylated AM stimulated cAMP production in vitro, showing lessened acute hypotensive action and a prolonged plasma half-life in comparison with native AM peptide in vivo.

Big angiotensin-25: a novel glycosylated angiotensin-related peptide isolated from human urine.

The renin-angiotensin system (RAS), including angiotensin II (Ang II), plays an important role in the regulation of blood pressure and body fluid balance. Consequently, the RAS has emerged as a key target for treatment of kidney and cardiovascular disease. In a search for bioactive peptides using an antibody against the N-terminal portion of Ang II, we identified and characterized a novel angiotensin-related peptide from human urine as a major molecular form. We named the peptide Big angiotensin-25 (Bang-25) because it consists of 25 amino acids with a glycosyl chain and added cysteine. Bang-25 is rapidly cleaved by chymase to Ang II, but is resistant to cleavage by renin. The peptide is abundant in human urine and is present in a wide range of organs and tissues. In particular, immunostaining of Bang-25 in the kidney is specifically localized to podocytes. Although the physiological function of Bang-25 remains uncertain, our findings suggest it is processed from angiotensinogen and may represent an alternative, renin-independent path for Ang II synthesis in tissue.

Functions of third extracellular loop and helix 8 of Family B GPCRs complexed with RAMPs and characteristics of their receptor trafficking.

At least one of three receptor activity-modifying proteins (RAMP1, RAMP2 and RAMP3) can interact with 10 G protein-coupled receptors (GPCRs; nine Family B GPCRs and a Family C GPCR). All three RAMPs interact with the calcitonin (CT) receptor (CTR), the CTR-like receptor (CLR), the vasoactive intestinal peptide (VIP)/pituitary adenylate cyclase-activating polypeptide (PACAP) 1 (VPAC1) and the VPAC2 receptor, which are all Family B GPCRs. Three RAMPs enable CTR to function as three heterodimeric receptors for amylin, which is a feeding suppression peptide. These RAMPs also transport the CLR to the cell surface, where they function as a CT gene-related peptide (CGRP) receptor (CLR/RAMP1 heterodimer) and two adrenomedullin (AM) receptors (CLR/RAMP2 and CLR/RAMP3 heterodimers). CGRP and AM are potent hypotensive peptides that exert powerful protective effects against multi-organ damage. We recently reported that the third extracellular loop (ECL3) of CLR governs the activation of AM, but not CGRP, signaling in the three CLR/RAMP heterodimers. Furthermore, we showed that in the presence of RAMP2, the eighth helix (helix 8) in the proximal portion of the cytoplasmic C-terminal tail of the CLR, which is thought to be present in all family B GPCRs, participates in receptor signaling. In addition, we demonstrated that overexpression of GPCR kinase (GRK) 2, GRK3 and GRK4 enhances the AM-induced internalization of the CLR/RAMP2 heterodimer. In this review, we describe these studies and consider their implications for other Family B GPCRs that can interact with RAMPs.

The third extracellular loop of the human calcitonin receptor-like receptor is crucial for the activation of adrenomedullin signalling.

BACKGROUND AND PURPOSE: The extracellular loops (ECLs) in Family A GPCRs are important for ligand binding and receptor activation, but little is known about the function of Family B GPCR ECLs, especially ECL3. Calcitonin receptor-like receptor (CLR), a Family B GPCR, functions as a calcitonin gene-related peptide (CGRP) and an adrenomedullin (AM) receptor in association with three receptor activity-modifying proteins (RAMPs). Here, we examined the function of the ECL3 of human CLR within the CGRP and AM receptors. EXPERIMENTAL APPROACH: A CLR ECL3 chimera, in which the ECL3 of CLR was substituted with that of VPAC2 (a Family B GPCR that is unable to interact with RAMPs), and CLR ECL3 point mutants were constructed and transiently transfected into HEK-293 cells along with each RAMP. Cell-surface expression of each receptor complex was then measured by flow cytometry; [(125) I]-CGRP and [(125) I]-AM binding and intracellular cAMP accumulation were also measured. KEY RESULTS: Co-expression of the CLR ECL3 chimera with RAMP2 or RAMP3 led to significant reductions in the induction of cAMP signalling by AM, but CGRP signalling was barely affected, despite normal cell-surface expression of the receptors and normal [(125) I]-AM binding. The chimera had significantly decreased AM, but not CGRP, responses in the presence of RAMP1. Not all CLR ECL3 mutants supported these findings. CONCLUSIONS AND IMPLICATIONS: The human CLR ECL3 is crucial for AM-induced cAMP responses via three CLR/RAMP heterodimers, and activation of these heterodimers probably relies on AM-induced conformational changes. This study provides a clue to the molecular basis of the activation of RAMP-based Family B GPCRs.

Adrenomedullin production is increased in colorectal adenocarcinomas; its relation to matrix metalloproteinase-9.

Adrenomedullin (AM) is highly expressed in various cancer cell lines, suggesting a possible association with cancer growth. In the present study, we examined the expression and/or concentration of AM, its related peptide, adrenomedullin2/intermedin (AM2/IMD) and their receptors in human colorectal cancer and the surrounding normal tissue. In addition, we assessed the correlation between the expression of AM and AM2/IMD with that of vascular endothelial growth factor (VEGF)-A and matrix metalloproteinase (MMP)-9. Using a specific immunoradiometric assay, we found that AM concentrations were 2-11-fold higher in colorectal cancer tissues than in the surrounding normal tissues. Moreover, real-time quantitative RT-PCR showed that the expression levels of preproAM (+548%), preproAM2/IMD (+2674%), calcitonin receptor-like receptor (CLR) (+518%), receptor activity modifying protein (RAMP)2 (+281%), RAMP3 (+178%), VEGF-A (+277%) and MMP-9 (+864%) mRNAs were significantly higher in cancer tissues than in the surrounding normal tissues, and there was a positive correlation between the gene expressions of MMP-9 and preproAM (r=0.352; p=0.005), but not with preproAM2/IMD (r=0.041, p=0.406). Both AM and AM2/IMD immunoreactivity were detected mainly within cancer cells, whereas MMP-9 immunoreactivity was mostly seen in the surrounding stroma. These findings suggest that AM produced in colorectal tumors acts in concert with MMP-9 in the stroma to contribute to the pathogenesis of colorectal cancer.

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 [(125)I]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 (100ng/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 Ser(449) to Ser(467) 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.

[Bone and bone related biochemical examinations. Hormone and hormone related substances. Calcitonin gene-related peptide (CGRP)].

Calcitonin gene-related peptide (CGRP) is secreted from osteoclasts and several cancer cells, as well as from sensory nerve endings innervating blood vessels and bones. A potent vasodilator, CGRP powerfully counteracts hypertensive responses and protects organs from blood pressure-related damage, though it also can precipitate migraine attacks, bronchoconstriction and hypersensitive colon. Because plasma CGRP concentrations are very low, even in patients, it is thought that CGRP functions as local modulator rather than as a circulating hormone. Nevertheless, it is useful to measure plasma CGRP when evaluating the severity of target diseases (e.g., heart failure and sepsis) and when screening or following up patients with neuroendocrine tumors (e.g., medullary thyroid carcinoma). That said, one should understand the biochemical characteristics of CGRP prior to blood sampling, and should use highly sensitive assays that detect only the active form of the molecule.

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.

Beyond vasodilation: the antioxidant effect of adrenomedullin in Dahl salt-sensitive rat aorta.

We have investigated the antioxidant effect of adrenomedullin (AM) on endothelial function in the Dahl salt-sensitive (DS) rat hypertension model. Dahl salt-resistant (DR) and DS rats were fed an 8% NaCl diet. In addition, the DS rats were subcutaneously infused with either saline or recombinant human AM for 4 weeks. Although systolic blood pressures measured weekly in AM- and saline-infused rats did not significantly differ, aortic O2*- levels were significantly (P<0.01) higher in the latter. Likewise, both endothelial nitric oxide synthase (eNOS) mRNA and protein were significantly higher in saline-infused DS rats. Infusion of AM reduced both O2*- and eNOS expression to levels comparable to those seen in DR rats. AM infusion also upregulated the gene expression of guanosine-5'-triphosphate cyclohydrolase I and downregulated the expression of p22(phox), suggesting that AM increased the NOS coupling and bioavailability of NO. AM possesses significant antioxidant properties that improve endothelial function.

Tumor necrosis factor-alpha downregulates adrenomedullin receptors in human coronary artery smooth muscle cells.

We examined the effects of tumor necrosis factor (TNF)-alpha on the expression and functionality of adrenomedullin (AM) receptors in cultured human coronary artery smooth muscle cells. Analysis of real-time quantitative polymerase chain reactions showed that these cells abundantly express two AM receptors comprised of calcitonin receptor-like receptor (CRLR) and receptor activity-modifying protein 1 (RAMP1) or RAMP2. TNF-alpha induced time- and dose-dependent decreases in the expression of CRLR and RAMP1/2 mRNAs, thereby diminishing AM-evoked cAMP production. The suppression of these three mRNAs was unaffected by inhibiting NOS, protein kinase G, protein kinase A, superoxide formation or NF-kappaB activation.

Effects of C-reactive protein on atherogenic mediators and adrenomedullin in human coronary artery endothelial and smooth muscle cells.

We examined the effects of recombinant human C-reactive protein (rhCRP) on atherosclerosis-related factors in cultured human coronary artery endothelial and smooth muscle cells (HCAECs and HCASMCs). After removing endotoxin from commercial rhCRP preparations using the appropriate column, the purified (P)-rhCRP retained the ability to Ca(2+)-dependently bind to phosphorylcholine, but did not augment the secretion of interleukin-6 and MCP-1 from HCAECs, as non-purified (NP)-rhCRP did. By contrast, P-rhCRP elicited 2- to 3-fold increases in the secretion of both hormones from HCASMCs, though the effect was smaller than that obtained with NP-rhCRP. Production of PAI-1 and endothelin-1 was little affected by either rhCRP preparation in either cell type. In addition, P-rhCRP dose-dependently diminished adrenomedullin release from both cell types, but did not affect adrenomedullin receptor expression or function. Our findings highlight the importance of removing endotoxin from commercial rCRP preparations and show that hCRP elicits atherogenic responses from HCASMCs, but not HCAECs.

Characterization of the human calcitonin gene-related peptide receptor subtypes associated with receptor activity-modifying proteins.

Coexpression of receptor activity-modifying proteins (RAMPs) with calcitonin receptor 2 (CTR2) or calcitonin receptor-like receptor (CRLR) leads to the formation of four functional heterodimeric receptors for human calcitonin gene-related peptide (hCGRP). In this study, we transfected hCGRP receptors into human embryonic kidney 293 cells and examined their pharmacological profiles using three dominant-negative (DN) RAMP mutants and various hCGRPalpha analogs. Fluorescence-activated cell-sorting analysis revealed that their cotransfection with CTR2 induced cell surface expression of all three RAMPs, and the three CTR2/RAMP heterodimers mediated equivalent levels of cAMP production in response to hCGRPalpha that were approximately 50-fold greater than were seen with CTR2 alone. By contrast, [Tyr0]hCGRPalpha binding and signaling were markedly weaker with CTR2/RAMP2 or -3 than with CTR2/RAMP1 or CRLR/RAMP1; likewise, 125I-[His10]hCGRPalpha bound most potently to CTR2/RAMP1. When CTR2 was coexpressed with DN RAMP1 or -2, hCGRPalpha-evoked responses were similar to those seen with CTR2 alone, despite the expression of both CTR2 and DN RAMP at the cell surface. But coexpression of DN RAMP3 with CTR2 significantly diminished hCGRPalpha signaling compared with that seen with CTR2 alone, indicating that DN RAMP3 is able to function as a negative regulator of CTR2 function. Competition experiments showed the relative agonist sensitivity of the four receptors to be hCGRPalpha > [Tyr0]hCGRPalpha > [Cys(Et)2,7]hCGRPalpha > [Cys(ACM)2,7]hCGRPalpha. Of the linear analogs, [Cys(ACM)2,7]hCGRPalpha (ACM, acetylmethoxy) enhanced cAMP formation only via CTR2/RAMP1, whereas [Cys(Et2,7)]hCGRPalpha acted via CRLR/RAMP1 and somewhat less potently via CTR2/RAMP1. Thus, among the three CGRP8-37-insensitive receptors, CTR2/RAMP1 is most sensitive to the two linear analogs, suggesting that it could be classified as a CGRP2 receptor. Moreover, the combined use of iodinated CGRPalpha analogs may be useful for defining the CGRP1 receptor.

Chronic salt loading upregulates expression of adrenomedullin and its receptors in adrenal glands and kidneys of the rat.

The vasodilator peptide adrenomedullin (AM) elicits diuresis and natriuresis and inhibits aldosterone secretion. The aim of this study was to better understand the role of AM in maintaining water and electrolyte balance during chronic salt loading. Male Wistar rats were divided into a high salt (HS) group that received a diet containing 8% sodium chloride (NaCl) and a normal salt group that received a diet containing 0.4% NaCl. Plasma AM concentrations as well as expression of AM mRNA in the adrenal gland and kidney were then measured after 3, 7, 14, and 28 days. After 28 days, sodium and water excretion were significantly higher in HS rats than in control, although blood pressure and fluid volume were not significantly affected. Moreover, although plasma AM remained unchanged for up to 14 days, it was increased 2.5-fold in HS rats after 28 days on a high salt diet, and there were corresponding 3-fold and 1.5-fold increases in the levels of AM mRNA in the adrenal gland and kidney, respectively. At the same time, expression of calcitonin receptor-like receptor mRNA was significantly upregulated in both kidney and adrenal gland, as was expression of receptor activity-modify protein 1 (RAMP1) and RAMP2 mRNA in the adrenals and expression of RAMP3 in kidneys. Taken together, these results suggest that AM plays a role in the regulation of water and electrolyte balance in animals chronically ingesting high levels of salt.

Angiotensin II modulates gene expression of adrenomedullin receptor components in rat cardiomyocytes.

Both adrenomedullin (AM) and angiotensin II (Ang II) are locally-acting hormones in the cardiac ventricles. Previously we reported that AM inhibits Ang II-induced hypertrophy of cultured rat neonatal cardiomyocytes. In this study, we examined whether Ang II affects the gene expression of the AM receptor components of calcitonin-receptor-like receptor (CRLR) and receptor-activity-modifying protein (RAMP) in rat cardiomyocytes. The mRNA levels of RAMP1 and RAMP3 were significantly elevated following 24-h treatment with Ang II without a change of those of RAMP2 and CRLR. AM increased the intracellular cAMP level and the cAMP accumulation by AM was significantly amplified by the 24-h preincubation with Ang II. The effects of Ang II on RAMP1 and RAMP3 expression were abolished by an Ang II type 1 (AT1) receptor antagonist, but not by an AT2 receptor antagonist. Thus, Ang II modulates gene expression of the AM receptor components via AT1 receptor, suggesting alteration of AM actions by Ang II in cultured rat cardiomyocytes.

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.

Expression of adrenomedullin and its receptor by chondrocyte phenotype cells.

For clarifying a process of de-differentiation in culturing chondrocytes, the present study was undertaken to investigate the secretion of adrenomedullin (AM) by chondrocyte phenotype cells and whether or not AM effects this proliferation in a cAMP-dependent fashion. Chondrocyte phenotype cells expressed AM and the AM receptor, and secreted high concentration of AM into the culture medium. When added to cultures, AM increased the intracellular cAMP level and decreased the number of these cells in a similar concentration-dependent fashion. Addition of forskolin and dibutyryl-cAMP caused a significant decrease in the number of these cells. Furthermore, the effect of AM was inhibited by a cAMP-dependent protein kinase A inhibitor (H89). The present findings indicate that AM has an autocrine/paracrine type of anti-proliferative effect on these cells mediated via a cAMP-dependent pathway and raise the possibility that AM plays a role in the local modulation of a process of de-differentiation by culturing chondrocyte phenotype cells.