Pro Region Research Articles

CDNA sequence analysis of seven peptide toxins from the spider Selenocosmia huwena

Seven cDNAs encoding six toxins HWTX-I, HWTX-II, HWTX-IIIa, HWTX-IV, HWTX-V, HWTX-VII and one lectin SHL-I, from the spider Selenocosmia huwena, were cloned and sequenced. On the basis of their amino acid sequences, we designed and synthesized 3′ RACE and 5′ RACE primer. By overlapping the two partial cDNA sequences obtained by 3′ and 5′ RACE, their full-length cDNA sequences were obtained. All of the cDNAs of these seven peptides encode a precursor including a potential signal peptide of 21–24 residues, a mature toxin of about 30 residues and an intervening pro region. The prepro regions of HWTX-I, HWTX-IIIa, HWTX-IV, HWTX-V and SHL-I were demonstrated, by the comparison of the cDNA sequences, to have high similarity, which is concert with the similar inhibitor cystine knot motif of HWTX-I, HWTX-IV and SHL-I although their functions are different. It was also demonstrated that, HWTX-II and HWTX-VII share the highly similar prepro region which is different from that of HWTX-I, HWTX-IV and SHL-I. The three dimensional structure of HWTX-II has been determined to exhibit a different motif. This indicates that the seven peptides from S. huwena could be classified into two different superfamilies according to the prepro region of cDNA sequences.

Interdependent Folding of the N- and C-Terminal Domains Defines the Cooperative Folding of α-Lytic Protease

Alpha-lytic protease (alphaLP) serves as an important model in achieving a quantitative and physical understanding of protein folding reactions. Synthesized as a pro-protease, alphaLP belongs to an interesting class of proteins that require pro regions to facilitate their proper folding. alphaLP's pro region (Pro) acts as a potent folding catalyst for the protease, accelerating alphaLP folding to its native conformation nearly 10(10)-fold. Structural and mutational studies suggested that Pro's considerable foldase activity is directed toward structuring the alphaLP C-terminal domain (CalphaLP), a seemingly folding-impaired domain, which is believed to contribute significantly to the high-energy folding and unfolding transition states of alphaLP. Pro-mediated nucleation of alphaLP folding within CalphaLP was hypothesized to subsequently enable the alphaLP N-terminal domain (NalphaLP) to dock and fold, completing the formation of native protease. In this paper, we find that ternary folding reactions of Pro and noncovalent NalphaLP and CalphaLP domains are unaffected by the order in which the components are added or by the relative concentrations of the alphaLP domains, indicating that neither discrete CalphaLP structuring nor docking of the two alphaLP domains is involved in the folding transition state. Instead, the rate-limiting step of these folding reactions appears to be a slow and concerted rearrangement of the NalphaLP and CalphaLP domains to form active protease. This cooperative and interdependent folding of both protease domains defines the large alphaLP folding barrier and is an apparent extension of the highly cooperative alphaLP unfolding transition that imparts the protease with remarkable kinetic stability and functional longevity.

Effect of Ingested Nutrients on the Release of Thrittene into the Human Circulation

Thrittene is a recently described peptide with a sequence homologous with somatostatin-28 ((1-13)) but is produced independent of the preprosomatostatin gene. It is localized in epithelial cells in stomach and gut mucosal crypts and in neuronal cell bodies in the myenteric plexus and enteric axons. It is also present in human plasma. The aim of this study was to determine whether the release of thrittene into the circulation was affected by the ingestion of nutrients and, if so, whether the pattern of release was distinct from the closely related peptide somatostatin-28 (S-28). Thrittene was indirectly measured in human plasma by an RIA using antiserum F4. F4 interacts with the Asn(5)-Pro(6) region shared by S-28 and thrittene. The contribution of S-28 to F4 immunoreactivity (F4-IR) was determined using a specific two-site assay, and this measure was subtracted from the total F4-IR to give an estimate of thrittene levels. Plasma for assay was taken from healthy men on 4 separate days before and after intake of: a mixed meal (715 kcal), and meals containing primarily fat (25 g; 225 kcal), carbohydrate (100 g; 454 kcal), and protein (22 g; 100 kcal). After the mixed meal, both S-28 and thrittene rose by 50-100% within 30 min and gradually declined by 4 h. These increments were mimicked after ingestion of fat. By contrast, thrittene levels increased after carbohydrate but not protein intake, whereas S-28 concentrations rose after protein but not carbohydrate ingestion. These findings indicate that thrittene is secreted into the mammalian circulation during food intake and raise the possibility that thrittene may play a role in nutrient disposition. The dichotomy in responses of thrittene and S-28 to ingestion of the major macronutrients suggests that they are secreted from different gastrointestinal cells.

Structure of human pro-chymase: a model for the activating transition of granule-associated proteases.

Human chymase is a protease involved in physiological processes ranging from inflammation to hypertension. As are all proteases of the trypsin fold, chymase is synthesized as an inactive "zymogen" with an N-terminal pro region that prevents the transition of the zymogen to an activated conformation. The 1.8 A structure of pro-chymase, reported here, is the first zymogen with a dipeptide pro region (glycine-glutamate) to be characterized at atomic resolution. Three segments of the pro-chymase structure differ from that of the activated enzyme: the N-terminus (Gly14-Gly19), the autolysis loop (Gly142-Thr154), and the 180s loop (Pro185A-Asp194). The four N-terminal residues (Gly14-Glu15-Ile16-Ile17) are disordered. The autolysis loop occupies a position up to 10 A closer to the active site than is seen in the activated enzyme, thereby forming a hydrogen bond with the catalytic residue Ser195 and occluding the S1' binding pocket. Nevertheless, the catalytic triad (Asp102-His57-Ser195) is arrayed in a geometry close to that seen in activated chymase (all atom rmsd of 0.52 A). The 180s loop of pro-chymase is, on average, 4 A removed from its conformation in the activated enzyme. This conformation disconnects the oxyanion hole (the amides of Gly193 and Ser195) from the active site and positions only approximately 35% of the S1-S3 binding pockets in the active conformation. The backbone of residue Asp194 is rotated 180 degrees when compared to its conformation in the activated enzyme, allowing a hydrogen bond between the main-chain amide of residue Trp141 and the carboxylate of Asp194. The side chains of residues Phe191 and Lys192 of pro-chymase fill the Ile16 binding pocket and the base of the S1 binding pocket, respectively. The zymogen positioning of both the 180s and autolysis loops are synergistic structural elements that appear to prevent premature proteolysis by chymase and, quite possibly, by other dipeptide zymogens.

Rabbit endogenous retrovirus-H encodes a functional protease.

Recent studies have revealed that 'human retrovirus-5' sequences found in human samples belong to a rabbit endogenous retrovirus family named RERV-H. A part of the gag-pro region of the RERV-H genome was amplified by PCR from DNA in human samples and several forms of RERV-H protease were expressed in bacteria. The RERV-H protease was able to cleave itself from a precursor protein and was also able to cleave the RERV-H Gag polyprotein precursor in vitro whereas a form of the protease with a mutation engineered into the active site was inactive. Potential N- and C-terminal autocleavage sites were characterized. The RERV-H protease was sensitive to pepstatin A, showing it to be an aspartic protease. Moreover, it was strongly inhibited by PYVPheStaAMT, a pseudopeptide inhibitor specific for Mason-Pfizer monkey virus and avian myeloblastosis-associated virus. A structural model of the RERV-H protease was constructed that, together with the activity data, confirms that this is a retroviral aspartic protease.

Control by A-factor of a metalloendopeptidase gene involved in aerial mycelium formation in Streptomyces griseus.

In Streptomyces griseus, A-factor (2-isocapryloyl-3R-hydroxymethyl-gamma-butyrolactone) switches on aerial mycelium formation and secondary metabolite biosynthesis. An A-factor-dependent transcriptional activator, AdpA, activates multiple genes required for morphological development and secondary metabolism in a programmed manner. A region upstream of a zinc-containing metalloendopeptidase gene (sgmA) was found among the DNA fragments that had been isolated as AdpA-binding sites. The primary product of sgmA consisted of N-terminal pre, N-terminal pro, mature, and C-terminal pro regions. sgmA was transcribed in an AdpA-dependent manner, and its transcription was markedly enhanced at the timing of aerial mycelium formation. AdpA bound two sites in the region upstream of the sgmA promoter; one was at about nucleotide position -60 (A site) with respect to the transcriptional start point of sgmA, and the other was at about position -260 (B site), as determined by DNase I footprinting. Transcriptional analysis with mutated promoters showed that the A site was essential for the switching on of sgmA transcription and that the B site was necessary for the marked enhancement of transcription at the timing of aerial mycelium formation. Disruption of the chromosomal sgmA gene resulted in a delay in aerial hypha formation by half a day. SgmA is therefore suggested to be associated with the programmed morphological development of Streptomyces, in which this peptidase, perhaps together with other hydrolytic enzymes, plays a role in the degradation of proteins in substrate hyphae for reuse in aerial hypha formation.

Cloning, expression, and deletion analysis of large nanH of Clostridium perfringens ATCC 10543

The large sialidase gene ( nanH) of Clostridium perfringens ATCC 10543 was cloned in pCRII and subcloned in pQE70 expression vector. The large nanH consists of 2082 bp nucleotides and encodes 694 amino acids; large NanH is with four repeated “Asp-boxes” and one RIP (Arg–Ile–Pro) region formed by amino acids 266–268 in the sequence. It showed 26% of sequence homology with the small NanH from the same species (ATCC 10543). The large nanH, with six His residues adding to the C-terminus of NanH protein, was constructed in pQE70 (recombinant plasmid named pQE70-LSC) and expressed in Escherichia coli M15. The large NanH expressed in the periplasmic space of bacteria M15 showed 430-fold increase in large NanH enzymatic activity upon IPTG induction, as compared to the culture without IPTG added. The recombinant large NanH was purified with Ni–NTA and N-( p-aminophenyl) oxamic acid–agarose column chromatography. The purified enzyme showed specific activity of 136.6 U/mg when assayed with 4-MU-NeuAc as substrate. The truncated NanH (pQE70-Ld1), deletion of amino acids 1–215, exhibits similar specific activity, the same optimum temperature and optimum pH compared with those of wild-type large NanH.

Molecular cloning of HR1a and HR1b, high molecular hemorrhagic factors, from Trimeresurus flavoviridis venom

HR1a and HR1b are two high molecular weight (P-III class) hemorrhagic factors in the venom of Trimeresurus flavoviridis. In this study, we cloned cDNAs of the HR1a and HR1b precursors and analyzed their nucleotide sequences. The cDNA for HR1a was 2368 nucleotides in length and encoded an open reading frame (ORF) of 609 amino acids; that for HR1b was 2237 nucleotides and encoded an ORF of 614 amino acids. Both cDNAs belonged to the N-III class consisting of signal, pro, metalloproteinase, disintegrin-like and cysteine-rich regions, and shared strong amino acid sequence similarity (74.4%). The HR1b precursor was found to have an additional seven amino acid sequence at the carboxyl terminus compared with a mature form of HR1b.

The pro region N-terminal domain provides specific interactions required for catalysis of alpha-lytic protease folding.

The extracellular bacterial protease, alpha-lytic protease (alphaLP), is synthesized with a large, two-domain pro region (Pro) that catalyzes the folding of the protease to its native conformation. In the absence of its Pro folding catalyst, alphaLP encounters a very large folding barrier (DeltaG = 30 kcal mol(-1)) that effectively prevents the protease from folding (t(1/2) of folding = 1800 years). Although homology data, mutational studies, and structural analysis of the Pro.alphaLP complex suggested that the Pro C-terminal domain (Pro C-domain) serves as the minimum "foldase" unit responsible for folding catalysis, we find that the Pro N-terminal domain (Pro N-domain) is absolutely required for alphaLP folding. Detailed kinetic analysis of Pro N-domain point mutants and a complete N-domain deletion reveal that the Pro N-domain both provides direct interactions with alphaLP that stabilize the folding transition state and confers stability to the Pro C-domain. The Pro N- and C-domains make conflicting demands upon native alphaLP binding that are alleviated in the optimized interface of the folding transition state complex. From these studies, it appears that the extremely high alphaLP folding barrier necessitates the presence of both the Pro domains; however, alphaLP homologues with less demanding folding barriers may not require both domains, thus possibly explaining the wide variation in the pro region size of related pro-proteases.

Modulation of the electrostatic charge at the active site of foot-and-mouth-disease-virus leader proteinase, an unusual papain-like enzyme.

The leader proteinase (L(pro)) of foot-and-mouth-disease virus is an unusual papain-like cysteine proteinase. Synthesized without an N-terminal pro precursor region, it frees itself from the growing polypeptide chain by cleavage at its own C-terminus. It also possesses a unique electrostatic environment around the active site, essentially due to Asp(163), which orients the catalytic histidine residue, and Asp(164); the equivalent residues in papain are Asn(175) and Ser(176). The importance of these residues for L(pro) activity was examined by site-directed mutagenesis. Replacement of Asp(163) with asparagine reduced activity by five-fold towards a hexapeptide substrate and slightly delayed self-processing when expressed in rabbit reticulocyte lysates. However, no effect on the cleavage of the only known cellular substrate of L(pro), eukaryotic initiation factor 4GI (eIF4GI), was observed. In contrast, replacement of Asp(164) by either alanine, asparagine or lysine abrogated activity towards the hexapeptide. Furthermore, in all cases, the onset of both self-processing and eIF4GI cleavage were significantly delayed; the reaction rates were also diminished compared with those of the wild-type enzyme. The alanine-substituted enzyme was least affected, followed by those substituted with asparagine and lysine. The double mutant protein in which both aspartate residues were replaced by asparagine was most severely affected; it failed to complete either self-processing or eIF4GI cleavage within 3 h, compared with the 8 min required by the wild-type enzyme. Hence, we propose that the electrostatic charge of Asp(164), and to a lesser extent that of Asp(163), is extremely important for L(pro) to attain full activity upon synthesis.

Modulation of the electrostatic charge at the active site of foot-and-mouth-disease-virus leader proteinase, an unusual papain-like enzyme

The leader proteinase (Lpro) of foot-and-mouth-disease virus is an unusual papain-like cysteine proteinase. Synthesized without an N-terminal pro precursor region, it frees itself from the growing polypeptide chain by cleavage at its own C-terminus. It also possesses a unique electrostatic environment around the active site, essentially due to Asp163, which orients the catalytic histidine residue, and Asp164; the equivalent residues in papain are Asn175 and Ser176. The importance of these residues for Lpro activity was examined by site-directed mutagenesis. Replacement of Asp163 with asparagine reduced activity by five-fold towards a hexapeptide substrate and slightly delayed self-processing when expressed in rabbit reticulocyte lysates. However, no effect on the cleavage of the only known cellular substrate of Lpro, eukaryotic initiation factor 4GI (eIF4GI), was observed. In contrast, replacement of Asp164 by either alanine, asparagine or lysine abrogated activity towards the hexapeptide. Furthermore, in all cases, the onset of both self-processing and eIF4GI cleavage were significantly delayed; the reaction rates were also diminished compared with those of the wild-type enzyme. The alanine-substituted enzyme was least affected, followed by those substituted with asparagine and lysine. The double mutant protein in which both aspartate residues were replaced by asparagine was most severely affected; it failed to complete either self-processing or eIF4GI cleavage within 3 h, compared with the 8min required by the wild-type enzyme. Hence, we propose that the electrostatic charge of Asp164, and to a lesser extent that of Asp163, is extremely important for Lpro to attain full activity upon synthesis.

Utility of (His)6 tag for purification and refolding of proplasmepsin-2 and mutants with altered activation properties.

Plasmepsin-2 is a malarial aspartic proteinase that has been implicated in the initial steps of hemoglobin degradation in parasites and thus represents an attractive antimalarial target. Escherichia coli expressed proplasmepsin-2 is capable of activation at acidic pH by autocatalytic cleavage of the pro part region, which results in products of different length. We designed a 10-amino-acid deletion in the pro part region that allows faster generation of homogeneous enzyme upon activation. Incorporation of a (His)6 tag onto the N-terminus of the pro part enables on-column refolding of proplasmepsin-2 and simplifies proenzyme purification and pro part separation after activation. The proposed purification procedure results in highly pure and easily crystallizable enzyme.

Spectroscopic monitoring of local conformational changes during the intramolecular domain-domain interaction of the ryanodine receptor.

The amino (N)-terminal and central regions of the ryanodine receptor (RyR) containing most mutation sites of malignant hyperthermia (MH) and central core disease (CCD) seem to be involved in the Ca(2+) channel regulation. Our recent peptide probe study (Yamamoto, T., El-Hayek, R., and Ikemoto, N. (2000) J. Biol. Chem. 275, 11618-11625) suggested the hypothesis that a close contact between the N-terminal and central domains (zipping) stabilizes the closed-state of the channel, while removal of the contact (unzipping) deblocks the channel, causing channel-activation effects. We here report the results of our recent effort to monitor local conformational changes in the putative domain-domain interaction site to test this hypothesis. The conformation-sensitive fluorescence probe, methyl coumarin acetamide (MCA), was incorporated into RyR in a protein- and site-specific manner by using DP4 (the peptide corresponding to the Leu(2442)-Pro(2477) region of the central domain) as a site-directing carrier. The site of MCA labeling was localized in the 150 kDa N-terminal region of RyR, indicating that DP4 and its in vivo counterpart (a portion of the central domain) interact with the N-terminal region. RyR-activating domain peptides, DP4 and DP1 (corresponding to the Leu(590)-Cys(609) region of the N-terminal domain), and depolarization of the T-tubule moiety of the triad (physiologic stimulation) induced a rapid decrease in the fluorescence intensity of the protein-bound MCA and Ca(2+) release at a somewhat slower rate. The accessibility of the protein-bound MCA to the fluorescence quencher was increased in the presence of DP4. These results are all consistent with the above hypothesis.

The pro region of Toxoplasma ROP1 is a rhoptry-targeting signal

The rhoptries of Toxoplasma gondii are regulated secretory organelles involved in the invasion of host cells. Rhoptry proteins are synthesised as pre-pro-proteins that are processed first to pro-proteins upon entrance into the secretory pathway, then processed again to their mature forms late in the secretory pathway. The pro-mature processing site of the rhoptry protein ROP1 has been determined, paving the way for understanding the role of the pro region in rhoptry protein function. We demonstrate here that the ROP1 pro region is sufficient for targeting a reporter protein (amino acids 34–471 of the Trypanosoma brucei VSG117 protein) to the rhoptries. These results, together with our previous work showing that rhoptry targeting is unaffected by deletion of the pro region, indicate that the ROP1 protein contains at least two signals that can function in rhoptry targeting.

The Pro Domain of β-Secretase Does Not Confer Strict Zymogen-like Properties but Does Assist Proper Folding of the Protease Domain

beta-Secretase (BACE) is a membrane-bound aspartyl protease that cleaves the amyloid precursor protein to generate the N terminus of the amyloid beta peptide. BACE is expressed as a precursor protein containing Pre, Pro, protease, transmembrane, and cytosolic domains. A soluble BACE derivative (PreProBACE460) that is truncated between the protease and transmembrane domains was produced by baculovirus-mediated expression. ProBACE460 was purified from conditioned media of infected insect cells using immobilized concanavalin A and immobilized BACE inhibitor, P10-P4' Stat(Val). Furin cleaves ProBACE460 between the Pro and protease regions to generate mature BACE460. The k(cat)/K(m) of ProBACE460 when assayed with a polypeptide substrate is only 2.3-fold less than that of BACE460. This finding and the similar inhibitory potency of P10-P4' Stat(Val) for ProBACE460 and BACE460 suggest that the Pro domain has little effect on the BACE active site. Exposure of ProBACE460 to guanidine denaturation/renaturation results in a 7-fold higher recovery of BACE activity than when BACE460 is similarly treated. The presence of free BACE Pro peptide during renaturation of BACE460 but not ProBACE460 increases recovery of activity. These findings show that the Pro domain in ProBACE460 does not suppress activity as in a strict zymogen but does appear to facilitate proper folding of an active protease domain.

Human recombinant pro-dipeptidyl peptidase I (cathepsin C) can be activated by cathepsins L and S but not by autocatalytic processing.

Human dipeptidyl peptidase I was expressed in the insect cell/baculovirus system and purified in its active (rhDPPI) and precursor (pro-rhDPPI) forms. RhDPPI was very similar to the purified enzyme (hDPPI) with respect to glycosylation, enzymatic processing, oligomeric structure, CD spectra, and catalytic activity. The precursor, which was a dimer, could be activated approximately 2000-fold with papain. Cathepsin L efficiently activated pro-rhDPPI in vitro at pH 4.5 (k(app) approximately 2 x 10(3) min(-)(1) M(-)(1)), and two cleavage pathways were characterized. The initial cleavage was within the pro region between the residual pro part and the activation peptide. Subsequently, the activation peptide was cleaved from the catalytic region, and the latter was cleaved into the heavy and light chains. Alternatively, the pro region was first separated from the catalytic region. Cathepsin S was a less efficient activating enzyme. Cathepsin B and rhDPPI did not activate pro-rhDPPI, and the proenzyme was incapable of autoactivation. Incubation of both pro-rhDPPI and rhDPPI with cathepsin D resulted in degradation. Cystatin C and stefins A and B inhibited rhDPPI with K(i) values in the nanomolar range (K(i) = 0.5-1.1 nM). The results suggest that cathepsin L could be an important activator of DPPI in vivo and that cathepsin D and possibly the cystatins may contribute to DPPI downregulation.

Construction of cells expressing neurotrophins.

AbstractThe ability to introduce genes of neurotrophic factors into eukaryotic cells provides a novel way to study neurobiological processes in vitro and in vivo (1–6). To study the functional role of the growth factors in vivo, genetically modified cells expressing neurotrophin have been grafted in the central nervous system (CNS) (2,3,6). In different animal models of CNS degenerative diseases, neurotrophic factors secreted from grafted cells can prevent neuronal degeneration and promote functional recovery after injury (1–11). The neurotrophin family is comprised of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and neurotrophin-4/5 (NT-4/5) (12–14). Although structurally similar, they differ in their regional and temporal distribution, receptor specificity, and biological activity. NGF, the first prototypic neurotrophin, is synthesized as part of a large precursor protein within which mature, bioactive NGF occupies the C-terminal half, and the N-terminal half contains a signal peptide (18 amino acid) preceding a large pro region. The pro sequence in combination with the signal (pre) sequence is necessary for the secretion of correctly processed, biologically active NGF (15). All neurotrophins show considerable structural homology to NGF and contain similar signal peptide and pro sequences.KeywordsNerve Growth FactorHelper VirusProducer Cell LinePackaging Cell LineFresh Complete MediumThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

On the unfolding of α‐lytic protease and the role of the pro region

On the unfolding of α‐lytic protease and the role of the pro region

Proteolytic activation of recombinant pro-memapsin 2 (pro-beta-secretase) studied with new fluorogenic substrates.

Memapsin 2 (beta-secretase), a membrane-anchored aspartic protease, is involved in the cleavage of beta-amyloid precursor protein to form beta-amyloid peptide. The primary structure of memapsin 2 suggests that it is synthesized in vivo as pro-memapsin 2 and converted to memapsin 2 by an activating protease [Lin et al. (2000) Proc. Natl. Acad. Sci. U.S.A. 97, 1456-1460]. To simulate this activation mechanism and to produce stable mature memapsin 2 for kinetic/specificity studies, we have investigated the activation of recombinant pro-memapsin 2 by several proteases with trypsin-like specificity. Clostripain, kallikrein, and trypsin increased the activity of pro-memapsin 2. Clostripain activation was accompanied by the cleavage of the pro region to form mainly two activation products, Leu(30p)- and Gly(45p)-memapsin 2. Another activation product, Leu(28p)-memapsin 2, was also purified. Kinetics of the activated memapsin 2 were compared with pro-memapsin 2 using two new fluorogenic substrates, Arg-Glu(5-[(2-aminoethyl)amino]naphthalene-1-sulfonic acid (EDANS))-Glu-Val-Asn-Leu-Asp-Ala-Glu-Phe-Lys(4-(4-dimethylaminophe nyl azo)benzoic acid (DABCYL))-Arg and (7-methoxycoumarin-4-yl)acetyl (MCA))-Ser-Glu-Val-Asn-Leu-Asp-Ala-Glu-Phe-Lys(2,4-dinitrophenyl (DNP)). These results establish that the activity of pro-memapsin 2 stems from a part-time and reversible uncovering of its active site by its pro region. Proteolytic removal of part of the pro-peptide at Leu(28p) or Gly(45p), which diminishes the affinity of the shortened pro-peptide to the active site, results in activated memapsin 2. These results also suggest that Glu(33p)-memapsin 2 observed in the cells expressing this enzyme [Vassar et al. (1999) Science 286, 735-741; Yan et al. (1999) Nature 402, 533-537] is an active intermediate of in vivo activation, or that the peptide Glu(33p)-Arg(44p) may serve a regulatory role.

Functional Importance of the Ala116–Pro136 Region in the Calcium-sensing Receptor: CONSTITUTIVE ACTIVITY AND INVERSE AGONISM IN A FAMILY C G-PROTEIN-COUPLED RECEPTOR

The calcium-sensing receptor (CaR) belongs to family C of the G-protein-coupled receptor superfamily. To date 14 activating mutations in CaR showing increased sensitivity to Ca(2+) have been identified in humans with autosomal dominant hypocalcemia. Four of these activating mutations are found in the Ala(116)-Pro(136) region of CaR, indicating that this part of the receptor is particularly sensitive to mutation-induced activation. This region was subjected to random saturation mutagenesis, and 219 mutant receptor clones were isolated and screened pharmacologically in a high throughput screening assay. Selected mutants were characterized further in an inositol phosphate assay. The vast majority of the mutants tested displayed an increased affinity for Ca(2+). Furthermore, 21 of the mutants showed increased basal activity in the absence of agonist. This constitutive activity was not diminished when the mutations were transferred to a chimeric receptor Ca/1a consisting of the amino-terminal domain of the CaR and the 7 transmembrane and intracellular domains of the metabotropic glutamate receptor mGluR1a. CPCCOEt, a noncompetitive antagonist acting at the 7 transmembrane domain of mGluR1a, suppressed the elevated basal response of the constitutively activated Ca/1a mutants demonstrating inverse agonist activity of CPCCOEt. Taken together, our results demonstrate that the Ala(116)-Pro(136) region is of key importance for the maintenance of the inactive conformation of CaR.