Domains determining ligand specificity for Ca2+ receptors.

The Ca2+ receptor is a G protein-coupled receptor that enables parathyroid cells and certain other cells in the body to respond to changes in the level of extracellular Ca2+. The Ca2+ receptor is a member of a family of G protein-coupled receptors that includes metabotropic glutamate receptors (mGluRs), gamma-aminobutyric acidB receptors, and putative pheromone receptors. As a family, these receptors are characterized by limited sequence homology and an unusually large putative extracellular domain (ECD). The ECD of the mGluRs is believed to determine agonist selectivity, but the functions of the structural domains of the Ca2+ receptor are not known. To identify structural determinants for cation recognition and activation of the Ca2+ receptor (and to further study the mGluRs), two chimeric receptors were constructed in which the large ECD of the Ca2+ receptor and the mGluR1 were interchanged. When expressed in Xenopus laevis oocytes, one of these chimeras, named CaR/mGluR1 [ECD of the Ca2+ receptor and transmembrane domain (TMD) of the mGluR1], responded to cation agonists (Gd3+, Ca2+, neomycin) of the Ca2+ receptor at concentrations similar to those necessary for activation of the native Ca2+ receptor. A reciprocal construct, named mGluR1/CaR (ECD of the mGluR1 and TMD of the Ca2+ receptor), was responsive to mGluR agonists but was much less sensitive to two of three cation agonists known to activate the Ca2+ receptor. A deletion construct of the Ca2+ receptor (DeltantCaR), which lacked virtually the entire ECD, was only activated by one of three agonists tested. These results suggest that the primary determinants for agonist activation of both the Ca2+ receptor and the mGluRs are found in the large ECD and that the Ca2+ receptor is possibly distinguished from the mGluRs in that it may contain sites in the TMD that permit activation by certain cation agonists.

Novel insecticidal peptides from Tegenaria agrestis spider venom may have a direct effect on the insect central nervous system.

Fractionation of venom from an agelenid spider, Tegenaria agrestis, resulted in the isolation of a family of three peptides with potent insecticidal activity. These peptide toxins, TaITX-1, -2, and -3, whose sequences were revealed from cloned cDNAs, each consist of 50 amino acid residues, six of which are cysteines. They appear to be amidated at their C-termini and exhibit greater than 90% sequence identity. Unlike other reported spider toxins, the TaI toxins are processed from precursors containing no propeptide sequences. In lepidopteran larvae and corn rootworm beetles, the insecticidal Tegenaria toxins caused an unusual excitatory symptomatology with 50% paralytic doses ranging from 0.23 to 2.6 nmol/g. In a series of electrophysiological experiments performed in house fly larvae, these toxins caused an elevated rate of firing from central nervous system neurons. No significant effects were found when any peripheral sensory or motor systems were examined. Thus, it appears that the TaI toxins may act in a fashion not previously reported for insecticidal peptide toxins; they may act directly on the insect central nervous system.

N-linked glycosylation of the human Ca2+ receptor is essential for its expression at the cell surface.

The human Ca2+ receptor (hCaR) is a member of the superfamily of G protein-coupled receptors. Its large (approximately 600 residue) amino-terminal extracellular domain contains 9 potential N-linked glycosylation sites. Immunoblot of cell membranes derived from HEK-293 cells, stably transfected with the hCaR, showed two major immunoreactive bands of approximately 150 and 130 kDa, respectively. Complete digestion of the membranes with PN-glycosidase F yielded a single major immunoreactive band of approximately 115 kDa, confirming the presence of N-linked glycosylation. Treatment of these cells with tunicamycin, which blocks N-linked glycosylation, inhibited signal transduction in response to Ca2+. Flow cytometric analysis showed decreased expression of the hCaR on the cell membrane in tunicamycin-treated cells. Immunoblot of tunicamycin-treated cells showed a reduction in the amount of the 150-kDa band and conversion of the 130-kDa band to the presumptively nonglycosylated 115-kDa form. Tunicamycin treatment of cells, transfected with a mutant hCaR complementary DNA containing a nonsense codon at position 599 preceding the 1st transmembrane domain, blocked the secretion of a 95-kDa protein, representing the amino-terminal extracellular domain, into the medium. These results demonstrate that N-linked glycosylation is required for normal expression of the hCaR at the cell surface.

Clustered inactivating mutations and benign polymorphisms of the calcium receptor gene in familial benign hypocalciuric hypercalcemia suggest receptor functional domains.

The predominant variety of familial benign hypocalciuric hypercalcemia (FBHH) is FBHH(3q), which is associated with presumed inactivating mutations of the cell surface calcium receptor (CaR) gene on chromosome 3q13.3-q21. We sought mutations of the CaR gene in FBHH by direct sequencing of PCR-amplified genomic DNA from 14 affected families: 8 mapped to 3q13, 1 mapped to chromosome 19p, and 5 unmapped. We sequenced the entire coding region of the gene (exons 2-7) in one or two affected members of each family and found six point mutations that altered one amino acid, cosegregated with hypercalcemia, and were absent in more than 100 unaffected persons. Four mutations were unique (S53P, D215G, S657Y, and P748R), and two had been reported previously (P55L and R185Q). Of four mutant CaR proteins expressed in Xenopus oocytes, three were deficient in extracellular Ca2+-induced signaling. No CaR mutations were found in eight families, including the one mapped to chromosome 19p. Three benign polymorphisms occurred in the COOH-terminal region of the CaR protein in 10%, 15%, and 30% of more than 100 unaffected persons. Thus, FBHH-causing CaR mutations were clustered in the NH2-terminal extracellular and membrane-spanning regions of the receptor protein. We suggest that these are important functional domains, probably for calcium binding and signal transduction, respectively. Finally, mutations in regulatory or intronic regions of the CaR gene may also underlie many cases of FBHH.

Characterization and cloning of insecticidal peptides from the primitive weaving spider Diguetia canities.

Three potent insecticidal peptide toxins were purified from the venom of the primitive weaving spider, Diguetia canities. The toxins share significant homology (> 40%) in their amino acid sequences and are of related size (masses of 6371-7080 Da). In lepidopteran larvae, the toxins cause a progressive spastic paralysis, with 50% paralytic doses (PD50S) ranging from 0.38 to 3.18 nmol/g, suggesting them to be among the most potent insecticidal compounds yet described from arthropod venoms. The most potent of these toxins, DTX9.2, was cloned using a reverse transcription-polymerase chain reaction (RT-PCR). The cDNA encodes a 94 amino acid precursor which is processed to the active 56 amino acid peptide by removal of a signal and propeptide sequence. The gene encoding DTX9.2 was isolated and characterized. The transcriptional unit spans 5.5 kilobases and is segregated into five exons. DNA sequences upstream from the first exon contain a TATA box and two palindromic sequences (one with homology to a CAAT consensus) which together may constitute a functional promoter. The highly segmented gene structure observed for this small peptide suggests that a mechanism such as exon shuffling may have played a role in the evolution of this toxin family.

Crystallization of the MS2 translational repressor alone and complexed to bromouridine.

The coat protein from the MS2 bacteriophage plays a dual role by encapsidating viral RNA and also by binding RNA as a translational repressor. In order to study the isolated dimer in a conformation not influenced by capsid interactions, a mutant molecule was crystallized that is defective in capsid assembly but is an active repressor. The unassembled dimer crystallized in the space group P21212 with a = 76.2, b = 55.7, and c = 28.4 A. In these crystals, monomers were related by twofold symmetry. When this dimer was co-crystallized with 5-bromouridine, crystals formed in space group R3 with a = b = 155.9 A, c = 29.9 A, gamma = 120 degrees; the dimer was the asymmetric unit.

Functional consequences of posttranslational isomerization of Ser46 in a calcium channel toxin.

The venom of the funnel-web spider Agelenopsis aperta contains several peptides that paralyze prey by blocking voltage-sensitive calcium channels. Two peptides, omega-Aga-IVB (IVB) and omega-Aga-IVC (IVC), have identical amino acid sequences, yet have opposite absolute configurations at serine 46. These toxins had similar selectivities for blocking voltage-sensitive calcium channel subtypes but different potencies for blocking P-type voltage-sensitive calcium channels in rat cerebellar Purkinje cells as well as calcium-45 influx into rat brain synaptosomes. An enzyme purified from venom converts IVC to IVB by isomerizing serine 46, which is present in the carboxyl-terminal tail, from the L to the D configuration. Unlike the carboxyl terminus of IVC, that of IVB was resistant to the major venom protease. These results show enzymatic activities in A. aperta venom being used in an unprecedented strategy for coproduction of necessary neurotoxins that possess enhanced stability and potency.