The history of parathyroid hormone and its receptor: structure-based design of parathyroid hormone analogues.
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Amino Acid Sequence↗
Biomedical subjects
H Jüppner
Publications and source records attributed to H Jüppner.
Cloning and characterization of the vitamin D receptor from Xenopus laevis.
The Vitamin D receptor (VDR), a member of the nuclear receptor superfamily, mediates the effects of 1,25-dihydroxyvitamin D3 on mineral ion homeostasis. Although the mammalian and avian VDRs have been extensively studied, little is known about the VDR in lower vertebrate species. To address this, we have isolated the Xenopus laevis VDR (xVDR) complementary DNA. Overall, the xVDR shares 79%, 73%, 73%, and 75% identity at the amino acid level with the chicken, mouse, rat, and human VDRs, respectively. The amino acid residues and subdomains important for DNA binding, hormone binding, dimerization, and transactivation are mostly conserved among all VDR species. The xVDR polypeptide can heterodimerize with the mouse retinoid X receptor alpha, bind to the rat osteocalcin vitamin D response element (VDRE), and induce vitamin D-dependent transactivation in transfected mammalian cells. Northern analysis reveals two xVDR messenger RNA species of 2.2 kb and 1.8 kb in stage 60 Xenopus tissues. In the adult, xVDR expression is detected in many tissues including kidney, intestine, skin, and bone. During Xenopus development, xVDR messenger RNA first appears at developmental stage 13 (pre-neurulation), increasing to maximum at stages 57-61 (metamorphosis). Our data demonstrate that, in Xenopus, VDR expression is developmentally regulated and that the vitamin D endocrine system is highly conserved during evolution.
Aging↗
Constitutive activation of the cyclic adenosine 3',5'-monophosphate signaling pathway by parathyroid hormone (PTH)/PTH-related peptide receptors mutated at the two loci for Jansen's metaphyseal chondrodysplasia.
Two different activating PTH/PTH-related peptide (PTHrP) receptor mutations, H223R and T410P, were recently identified as the most likely cause of Jansen's metaphyseal chondrodysplasia. To assess the functional importance of either amino acid position in the human PTH/PTHrP receptor, H223 and T410 were individually replaced by all other amino acids. At position 223, only arginine and lysine led to agonist-independent cAMP accumulation; all other amino acid substitutions resulted in receptor mutants that lacked constitutive activity or were uninformative due to poor cell surface expression. In contrast, most amino acid substitutions at position 410 conferred constitutive cAMP accumulation and affected PTH/PTHrP receptor expression not at all or only mildly. Mutations corresponding to the H223R or T410P exchange in the human PTH/PTHrP receptor also led to constitutive activity when introduced into the opossum receptor homolog, but showed little or no change in basal cAMP accumulation when introduced into the rat PTH/PTHrP receptor. The PTH/PTHrP receptor residues mutated in Jansen's disease are conserved in all mammalian members of this family of G protein-coupled receptors. However, when the equivalent of either the H223R or the T410P mutation was introduced into several other related receptors, including the PTH2 receptor and the receptors for calcitonin, secretin, GH-releasing hormone, glucagon-like peptide I, and CRH, the resulting mutants failed to induce constitutive activity. These studies suggest that two residues in the human PTH/PTHrP receptor, 223 and 410, have critical roles in signal transduction, but with different sequence constrains.
Amino Acid Sequence↗
Design, synthesis and utility of novel benzophenone-containing calcitonin analogs for photoaffinity labeling the calcitonin receptor.
Calcitonin (CT) is a 32-amino-acid calciotropic peptide hormone which acts on target cells via a G protein-coupled seven-transmembrane receptor (CTR). In this study, we report the design, synthesis and characterization of four potent bioactive and photoreactive CT analogs, each of which contains a single benzophenone moiety inserted at different and discrete locations within the CT molecule. Replacement of all Lys residues in salmon CT (sCT) with Arg, followed by replacement of hydrophobic residues with a Lys(epsilon-p-benzoylbenzoyl) residue [Lys(epsilon-pBz2)] was found to preserve high biological activity. We substituted Val8, Leu16 and Leu19 by Lys(epsilon-pBz2), and acylated the N-terminus by a pBz2 moiety, thus distributing the photoaffinity moiety in the different analogs across a large portion of the CT sequence. With both transfected and endogenous CTRs from several species, all four benzophenone-containing analogs were shown to be virtually indistinguishable from the parent sCT analog in both receptor binding properties and stimulation of cAMP accumulation. Upon photolysis, in the presence of CTR, the radioiodinated photoreactive CT analog ([Arg11,18,Lys19(epsilon-pBz2)]sCT (K19)) covalently labels a membrane component of approximately 70 kDa. Receptor cross-linking is inhibited specifically in the presence of excess sCT. We also examined the interaction of these CT analogs with a hemagglutinin (HA) epitope-tagged CTR. The HA-CTR displayed CT binding and CT-dependent cAMP stimulation identical with native CTR. Both K19 and another bioactive analog (-Arg11,18, Lys8(epsilon-pBz2)]sCT (K8)) specifically photoaffinity cross-link to the HA-CTR. These benzophenone-containing CT analogs should facilitate studies of hormone-receptor interactions and allow the direct identification of a CT binding domain(s) within the receptor by the analysis of photochemically cross-linked conjugates.
Amino Acid Sequence↗
Structure based design of parathyroid hormone analogs.
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Amino Acid Sequence↗
Full activation of chimeric receptors by hybrids between parathyroid hormone and calcitonin. Evidence for a common pattern of ligand-receptor interaction.
Calcitonin (CT) and parathyroid hormone (PTH), whose receptors belong to the same family of G protein-coupled receptors, share no amino acid sequence homology and selectively activate either CT or PTH receptors. We now show, however, that reciprocal hybrid ligands (CT/PTH and PTH/CT), which do not activate the "wild-type" receptors, activate PTH/CT and CT/PTH receptor chimeras, respectively. Our findings indicate that PTH and CT share a similar architecture with at least two functional, receptor-specific domains. These domains are sufficiently independent to permit synthetic hybrid ligands to efficiently activate appropriate receptor chimeras. Therefore, both ligands follow, despite their very different primary sequences, a common pattern of ligand-receptor interaction.
Animals↗
Constitutively activated receptors for parathyroid hormone and parathyroid hormone-related peptide in Jansen's metaphyseal chondrodysplasia.
BACKGROUND: An activating mutation of the receptor for parathyroid hormone (PTH) and parathyroid hormone-related peptide (PTHrP) was recently found in a patient with Jansens's metaphyseal chondrodysplasia, a rare form of short-limbed dwarfism associated with hypercalcemia and normal or low serum concentrations of the two hormones. To investigate this and other activating mutations and to refine the classification of this unusual disorder, we analyzed genomic DNA from six additional patients with Jansen's disease. METHODS: Exons encoding the PTH-PTHrP receptor were amplified by the polymerase chain reaction (PCR), and the products were analyzed by gel electrophoresis or direct nucleotide-sequence analysis. Nucleotide changes were confirmed by restriction-enzyme digestion of genomic DNA or the PCR products. RESULTS: The previously reported mutation, which changes a histidine at position 223 to arginine (H223R), was found in genomic DNA from three of the six patients but not in DNA from their healthy relatives or 45 unrelated normal subjects. A novel missense mutation that changes a threonine in the receptor's sixth membrane-spanning region to proline (T410P) was identified in another patient but not in 62 normal subjects. In two patients with radiologic evidence of Jansen's metaphyseal chondrodysplasia but less severe hypercalcemia, no receptor mutations were detected. In COS-7 cels expressing PTH-PTHrP receptors with the T410P or H223R mutation, basal cyclic AMP accumulation was four to six times higher than in cells expressing wild-type receptors. CONCLUSIONS: The expression of constitutively active PTH-PTHrp receptors in kidney, bone, and growth-plate chondrocytes provides a plausible genetic explanation for mineral-ion abnormalities and metaphyseal changes in patients with Jansen's disease.
Base Sequence↗
Converting parathyroid hormone-related peptide (PTHrP) into a potent PTH-2 receptor agonist.
Most of the bone and kidney-related functions of parathyroid hormone (PTH) and parathyroid hormone-related peptide (PTHrP) are thought to be mediated by the PTH/PTHrP receptor. Recently, a homologous receptor, the PTH-2 receptor, was obtained from rat and human brain cDNA libraries. This receptor displayed the remarkable property of responding potently to PTH, but not to PTHrP. To begin to define residues involved in the ligand specificity of the PTH-2 receptor, we studied the interaction of several PTH/PTHrP hybrid ligands and other related peptide analogs with the human PTH-2 receptor. The results showed that two sites in PTH and PTHrP fully account for the different potencies that the two ligands exhibited with PTH-2 receptors; residue 5 (His in PTHrP and Ile in PTH) determined signaling capability, while residue 23 (Phe in PTHrP and Trp in PTH) determined binding affinity. By changing these two residues of PTHrP to the corresponding residues of PTH, we were able to convert PTHrP into a ligand that avidly bound to the PTH-2 receptor and fully and potently stimulated cAMP formation. Changing residue 23 alone yielded [Trp23]hPTHrP-(1-36), which was an antagonist for the PTH-2 receptor, but a full agonist for the PTH/PTHrP receptor. Residues 5 and 23 in PTH and PTHrP thus play key roles in signaling and binding interactions, respectively, with the PTH-2 receptor. Receptor-selective agonists and antagonists derived from these studies could help to identify the biological role of the PTH-2 receptor and to map specific sites of ligand-receptor interaction.
Amino Acid Sequence↗
PTH/PTHrP receptor in early development and Indian hedgehog-regulated bone growth.
The PTH/PTHrP receptor binds to two ligands with distinct functions: the calcium-regulating hormone, parathyroid hormone (PTH), and the paracrine factor, PTH-related protein (PTHrP). Each ligand, in turn, is likely to activate more than one receptor. The functions of the PTH/PTHrP receptor were investigated by deletion of the murine gene by homologous recombination. Most PTH/PTHrP receptor (-/-) mutant mice died in mid-gestation, a phenotype not observed in PTHrP (-/-) mice, perhaps because of the effects of maternal PTHrP. Mice that survived exhibited accelerated differentiation of chondrocytes in bone, and their bones, grown in explant culture, were resistant to the effects of PTHrP and Sonic hedgehog. These results suggest that the PTH/PTHrP receptor mediates the effects of Indian Hedgehog and PTHrP on chondrocyte differentiation.
Animals↗
Down-regulation of the receptor for parathyroid hormone (PTH) and PTH-related peptide by PTH in primary fetal rat osteoblasts.
We studied the effects of parathyroid hormone (PTH) on PTH parathyroid hormone related peptide (PTHrP) receptor mRNA level, PTHrP binding and PTH-stimulated cyclic adenosine monophosphate (cAMP) accumulation in osteoblasts, derived from fetal rat calvariae (ROB). Cells isolated during 10-70 minutes of collagenase treatment were seeded at a density of 25,000 cells/cm2 and cultured for 4 days. These cells show a fast increase in cAMP production after stimulation for 5 minutes with 20 nM bovine parathyroid hormone(1-34) (bPTH(1-34)). When ROB are incubated with bPTH(1-34) (0.04-40nM) for 24 h, a dose-dependent decrease of the PTH/PTHrP receptor mRNA level, PTHrP binding, and PTH-stimulated cAMP accumulation can be observed. Pretreatment of ROB with a high concentration of bPTH(1-34) (40 nM) leads within 15 minutes to a decrease in PTH-stimulated cAMP accumulation. However, it takes > or = 3 h before a significant decrease in PTH/PTHrP receptor mRNA level can be observed. Also a significant decrease in PTHrP binding is observed after only 4 h of incubation with bPTH(1-34). Compared with bPTH(1-34), pretreatment of ROB with bPTH(3-34) (40 and 100 nM) for 24 h causes smaller decreases in PTH-stimulated cAMP accumulation, PTHrP binding, and in the PTH/PTHrP receptor mRNA level. We investigated the possible involvement of the protein kinase A signaling pathway in the regulation of the PTH/PTHrP receptor mRNA expression. Both forskolin and (Bu)2cAMP decreased PTHrP binding and PTH/PTHrP mRNA levels. These observations suggest that chronic activation of the PKA signaling pathway may down-regulate PTH/PTHrP receptor expression and thus hormone responsiveness in "normal" osteoblasts. In short, we found that the decrease of the PTH-stimulated cAMP accumulation after long-term pretreatment with bPTH(1-34) is correlated with both PTH/PTHrP receptor mRNA level and PTHrP binding. These data also suggest that the initial desensitization (< 30 minutes) of PTH-stimulated cAMP responsiveness by pretreatment with a high concentration of bPTH(1-34) (40 nM) is not dependent on the number of available PTH/PTHrP receptors. The protein kinase A signaling pathway is involved in the regulation of the PTH/PTHrP receptor, but, regarding the effect of bPTH(3-34), other signaling systems are also involved.
Animals↗
Receptors for parathyroid hormone and parathyroid hormone-related peptide: from molecular cloning to definition of diseases.
The parathyroid hormone/parathyroid hormone-related peptide receptor belongs to a distinct family of G protein-coupled receptors, the members of which usually signal through at least two second messenger systems, adenylate cyclase and phospholipase C. The parathyroid hormone/ parathyroid hormone-related peptide receptor is most abundantly expressed in bone, kidney and growth-plate chondrocytes, and, at lower levels, in a variety of fetal and adult tissues. To search for human diseases that are caused by parathyroid hormone/parathyroid hormone-related peptide receptor defects, genomic DNA of patients with pseudohypoparathyroidism type Ib and of patients with Jansen's metaphyseal chondrodysplasia was screened for mutations in all coding exons of the receptor gene. Inactivating parathyroid hormone/parathyroid hormone-related peptide receptor mutations were excluded in patients with pseudohypoparathyroidism type Ib. However, a receptor mutation that causes agonist-independent, constitutive cAMP accumulation was identified in a patient with Jansen's metaphyseal chondrodysplasia, a rare form of short-limbed dwarfism associated with hypercalcemia despite normal or low concentrations of parathyroid hormone and parathyroid hormone-related peptide. These findings allow the conclusion to be drawn that parathyroid hormone/parathyroid hormone-related peptide receptors mediate the endocrine actions of parathyroid hormone, which are required for the control of calcium homeostasis and the autocrine-paracrine actions of parathyroid hormone-related peptide, which are required for normal growth-plate development.
Adult↗
Cell-specific signal transduction of parathyroid hormone (PTH)-related protein through stably expressed recombinant PTH/PTHrP receptors in vascular smooth muscle cells.
PTH-related protein activates a G protein-coupled PTH/PTHrP receptor in many cell types and produces diverse biological actions. To study the signal transduction events associated with biological activity of the PTH/PTHrP receptor in vascular smooth muscle, a principal PTHrP-responsive tissue, rat aortic smooth muscle cells (A10) were stably transfected with a plasmid encoding a PTH/PTHrP receptor and tested for ligand binding, PTHrP-(1-34)-induced cAMP levels, inositol phosphate production, and cytosolic calcium transients. Of nineteen G418-resistant lines recovered, all exhibited high affinity binding [approximately dissociation constant (Kd) > 10(-10)) of iodinated [Tyr36]hPTHrP(1-36)NH2 and ligand-induced cAMP accumulation (2- to 100-fold), which was directly proportional to PTH/PTHrP receptor number (range 4 x 10(3) to 7 x 10(7) sites/cell]. PTHrP had no effect on intracellular calcium or inositol phosphate formation in any cell line regardless of receptor number despite the presence of detectable G alpha q). Transient overexpression of individual G alpha q proteins (G alpha q, G alpha 11 or G alpha 14) into PTH/PTHrP receptor-expressing A10 cells conferred the ability of PTHrP to increase intracellular calcium and inositol phosphate formation. Ligand activation of the recombinant PTH/PTHrP receptor elicited appropriate downstream biological effects in A10 cells including inhibition of DNA synthesis and osteopontin messenger RNA (mRNA) expression. Thus, a single PTH/PTHrP receptor, though capable of coupling to different G proteins, signals exclusively through a cAMP-dependent pathway in vascular smooth muscle.
Animals↗
Inverse agonism of amino-terminally truncated parathyroid hormone (PTH) and PTH-related peptide (PTHrP) analogs revealed with constitutively active mutant PTH/PTHrP receptors.
Inverse agonists, ligands that suppress spontaneous receptor signaling activity, have been described for a growing number of G protein-coupled receptors; however, none have been reported for the PTH/calcitonin/secretin receptor family. We took advantage of the constitutive signaling activity of two mutant forms of the PTH/PTH-related peptide (PTHrP) receptor, recently identified in patients with Jansen's metaphyseal chondrodysplasia, to screen for PTH and PTHrP analogs with inverse agonist activity. Two antagonist peptides, [Leu11, D-Trp12]hPTHrP(7-34)NH2 and [D-Trp12, Tyr34]bPTH-(7-34)NH2, displayed inverse agonist activity and reduced cAMP in COS-7 cells expressing either mutant receptor by 30-50% (EC50 approximately 50 nM). These data demonstrate that the concept of inverse agonism can be extended to this distinct family of G protein-coupled receptors and their cognate antagonist peptide ligands. Such ligands shall be useful probes of the multi-state conformational equilibria proposed for these receptors and could lead to new approaches for treating human diseases caused by receptor activating mutations.
Cell Line↗
Hypercalcemia due to constitutive activity of the parathyroid hormone (PTH)/PTH-related peptide receptor: comparison with primary hyperparathyroidism.
In Jansen's disease (JD), the hypercalcemia found in about half the cases is the result of a mutant, constitutively overactive, form of the PTH/PTHrP receptor, which in these cases also causes the skeletal dysplasia. The subject of the present report was first seen in 1956 and is still under treatment at the same medical center. We report the clinical course and a detailed study of calcium and bone metabolism carried out in 1976 and compare the results with those of six typical patients with mild primary hyperparathyroidism in whom exactly the same studies were carried out. In the patient with JD, the hypercalcemia was of early onset; chronic and nonprogressive; refractory to the administration of phosphate, glucocorticoid, and calcitonin; and accompanied by suppressed PTH levels as determined by two different immunoassays, an undetectable PTHrP level, increased excretion of nephrogenous cAMP (an in vivo bioassay of endogenous PTH production), decreased tubular reabsorption of phosphate, increased tubular reabsorption of calcium, increased biochemical indexes of bone turnover, and increased histological indexes of bone turnover on iliac bone histomorphometry after double tetracycline labeling. There was exaggerated loss of cortical bone and preservation of cancellous bone. All the results in JD relating to renal or skeletal effects of PTH excess were within or close to the ranges found in the hyperparathyroid patients, except that tubular reabsorption of phosphate was more depressed. Because PTH secretion was suppressed, any effects mediated by putative alternative receptors would have been diminished. We conclude that 1) the hypercalcemia due to constitutive overactivity of the PTH/PTHrP receptor is indistinguishable from that of mild primary hyperparathyroidism in clinical characteristics and renal tubular and skeletal features; and 2) the classic laboratory manifestations of primary hyperparathyroidism, with the possible exception of osteitis fibrosa cystica, can all be accounted for by overactivity of a single receptor.
Bone Diseases, Developmental↗
Truncation of the carboxyl-terminal region of the rat parathyroid hormone (PTH)/PTH-related peptide receptor enhances PTH stimulation of adenylyl cyclase but not phospholipase C.
The functional role of the rat parathyroid hormone(PTH)/PTH-related peptide (PTHrP) receptor's carboxyl-terminal region was characterized by comparing the binding and signaling properties of receptors that have 78 and 111 amino acid deletions (R513 and R480, respectively), with those of the 591-amino acid wild-type (WT) receptor. R480 and R513 have 4- and 1.5-fold lower apparent Kd values for rat PTH-(1-34) (rPTH), compared with the WT receptor (WT, 1.81 +/- 0.19 nM; R513, 1.24 +/- 0.12 nM; R480, 0.48 +/- 0.05 nM, mean +/- S.E.). PTH (100 nM)-stimulated cAMP accumulation and polyphosphoinositide hydrolysis both correlated positively with receptor expression. However, whereas PTH-stimulated polyphosphoinositide hydrolysis was indistinguishable among WT and either truncated mutant at comparable levels of expressed receptors, maximal PTH-stimulated cAMP accumulation was 4-6- and 2-3-fold higher in cells expressing R480 and R513, respectively. Furthermore, pretreatment of COS-7 cells with 100 ng/ml of pertussis toxin (PTX) enhanced PTH-stimulated cAMP accumulation in cells expressing the WT receptor, but failed to do so in cells expressing either R480 or R513. Thus, sequences in the PTH/PTHrP receptor's carboxyl-terminal tail lower the affinity of the WT receptor for agonist; directly interact with, or indirectly facilitate the interaction of the receptor with a PTX-sensitive G protein that inhibits adenylyl cyclase; and decrease the efficacy with which the receptor interacts with Gs.
Adenylate Cyclase Toxin↗
A constitutively active mutant PTH-PTHrP receptor in Jansen-type metaphyseal chondrodysplasia.
A single heterozygous nucleotide exchange in exon M2 of the gene encoding the parathyroid hormone-parathyroid hormone-related peptide (PTH-PTHrP) receptor was identified in a patient with Jansen-type metaphyseal chondrodysplasia, which changes a strictly conserved histidine residue at position 223 in the receptor's first intracellular loop to arginine. Constitutive, ligand-independent adenosine 3',5'-monophosphate accumulation was observed in COS-7 cells expressing the mutant PTH-PTHrP receptor but not in cells expressing the wild-type receptor. This finding explains the severe ligand-independent hypercalcemia and hypophosphatemia, and most likely the abnormal formation of endochondral bone, in this rare form of short-limbed dwarfism.
Amino Acid Sequence↗
Expression of parathyroid hormone-related peptide and its receptor messenger ribonucleic acid in human amnion and chorion-decidua: implications for secretion and function.
OBJECTIVE: Our purpose was to define the location and packaging of parathyroid hormone-related peptide in amnion-chorion and the potential target tissues for its action in fetal membranes. STUDY DESIGN: We studied fetal membranes by use of light microscopic immunocytochemistry with three monoclonal antibodies against distinct regions of the parathyroid hormone-related peptide molecule. For electron microscopy immunogold analysis with a monoclonal antibody specific to the 109-141 fragment was used to observe parathyroid hormone-related peptide intracellularly in amnion membrane and in the chorion layers. Multiplex reverse transcriptase-polymerase chain reaction with Southern blotting was used to identify parathyroid hormone/parathyroid hormone-related peptide receptor and control messenger ribonucleic acids in amnion and chorion-decidua. RESULTS: All monoclonal antibodies revealed immunoreactive parathyroid hormone-related peptide in the amniotic epithelial cells and in some fibroblast-like cells embedded in the extracellular matrix of the amnion. Parathyroid hormone-related peptide was also found in the chorion in fibroblast and trophoblast layers and in decidua. Ultrastructurally immunogold particles were evenly distributed throughout the amniotic epithelial cells and were present in apical microvilli and near the basal membranes. Electron microscopy studies of the chorion cytotrophoblast also showed freely dispersed immunogold particles of parathyroid hormone-related peptide with no packaging in secretory granules. Low to undetectable levels of parathyroid hormone/parathyroid hormone-related peptide receptor messenger ribonucleic acid were found in amnion tissue, whereas abundant receptor messenger ribonucleic acid was found in chorion-decidua. CONCLUSIONS: These results suggest the presence of a parathyroid hormone-related peptide paracrine system within the human fetal membranes.
Amnion↗
Functional properties of the PTH/PTHrP receptor.
The PTH/PTHrP receptor belongs to a novel family of G-protein-coupled receptors which also includes an insect receptor for a diuretic hormone and the protein encoded by a genomic DNA clone from Caenorhabditis elegans. Despite significant structural conservation, rat, opossum, and human PTH/PTHrP receptor homologs display distinct functional characteristics when tested with either [Arg2, Tyr34]hPTH(1-34)amide or [Nle8.18, Tyr34]bPTH(7-34)-amide. These PTH analogs, and chimeras between rat/opossum and between rat/human PTH/PTHrP receptors, led to the identification of receptor residues that appear to be involved in ligand/receptor interaction and receptor activation, respectively. The search for mutations in the PTH/PTHrP receptor gene in genomic DNA of patients with pseudohypoparathyroidism type Ib (PHP-Ib) revealed several silent polymorphisms and a missense mutation in the receptor's tail region which did not affect receptor function. Mutations in the PTH/PTHrP receptor are therefore rarely, if at all, responsible for PHP-Ib. A mutation in the PTH/PTHrP receptor is, however, the most likely cause of Jansen-type metaphyseal chondrodysplasia, a rare form of short-limbed dwarfism which is associated with severe hypercalcemia despite normal or low levels of circulating PTH and PTHrP. A missense mutation was identified which causes constitutive, ligand-independent receptor activation, and thus explains the laboratory and the growth-plate abnormalities in affected individuals.
Amino Acid Sequence↗