Expression, purification, and biochemical characterization of the amino-terminal extracellular domain of the human calcium receptor.

We purified the extracellular domain (ECD) of the human calcium receptor (hCaR) from the medium of HEK-293 cells stably transfected with a hCaR cDNA containing an isoleucine 599 nonsense mutation. A combination of lectin, anion exchange, and gel permeation chromatography yielded milligram quantities of >95% pure protein from 15 liters of starting culture medium. The purified ECD ran as an approximately 78-kDa protein on SDS-polyacrylamide gel electrophoresis and was found to be a disulfide-linked dimer. Its NH2-terminal sequence, carbohydrate content, and CD spectrum were defined. Tryptic proteolysis studies showed two major sites accessible to cleavage. These studies provide new insights into the structure of the hCaR ECD. Availability of purified ECD protein should permit further structural studies to help define the mechanism of Ca2+ activation of this G protein-coupled receptor.

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.

Monoclonal antibodies against synthetic peptides corresponding to the extracellular domain of the human Ca2+ receptor: characterization and use in studying concanavalin A inhibition.

We generated monoclonal antibodies against two synthetic peptides corresponding to residues 214-235 (ADD) and 374-391 (LRG) of the human Ca2+ receptor (hCaR) extracellular domain (ECD). Although both antibodies reacted well with their respective immunizing peptides on peptide-based enzyme linked immunosorbent assay, ADD was much more strongly reactive with the hCaR than LRG in assays such as immunoblots done under denaturing conditions. The opposite pattern was seen in flow cytometry analysis of the native receptor stably expressed in transfected 293 cells. We speculate that the ADD epitope is unexposed in the native receptor while the reverse is true for the LRG epitope. The ability to measure cell surface expression of the hCaR under native conditions using flow cytometry with the LRG monoclonal allowed us to study the basis for Concanavalin A (Con A) inhibition of CaR activation by Ca2+. Our studies show that Con A inhibition is partially accounted for by receptor internalization but, additionally, Con A may prevent Ca2+ stimulation directly by binding to carbohydrate residues in the receptor ECD.

Expression of G protein alpha-subunits in bovine parathyroid.

A G protein-coupled Ca(2+)-sensing receptor was recently cloned from bovine parathyroid and shown to mediate divalent cation regulation of PTH secretion. To define which G proteins might be coupled to the Ca(2+)-sensing receptor in parathyroid cells, we determined which G protein alpha-subunit messenger RNAs (mRNAs) are expressed in the parathyroid. We also considered the possibility that a novel parathyroid-specific G alpha might be present. We, therefore, used the reverse transcription-polymerase chain reaction to study the expression of G alpha subunits in a bovine parathyroid mRNA preparation. Degenerate primers, corresponding to two regions conserved in every G alpha subunit, the G3 and G4 sequences, were used to amplify G alpha complementary DNA fragments that were subcloned and sequenced. We found that mRNAs corresponding to G alpha s, G alpha i2, G alpha 11, G alpha 12, and G alpha z are the predominant G alpha mRNAs expressed in the bovine parathyroid. No novel G alpha mRNA was identified. Northern blots confirmed the expression of the cloned G alpha subunits and showed lower expression of G alpha o and G alpha i1 mRNAs. Immunoblots confirmed abundant expression of G alpha s, G alpha i2, and G alpha 11 and provided evidence for expression of G alpha i1 and G alpha i3, but not G alpha o. G alpha q mRNA was not identified by the degenerate primer reverse transcription-polymerase chain reaction strategy, but the immunoblot detected G alpha q protein, albeit at considerably lower levels than G alpha 11. The abundance of G alpha 11 relative to G alpha q in bovine parathyroid is consistent with but does not prove a role for G alpha 11 in coupling the Ca(2+)-sensing receptor to phospholipase C.

Two hexokinases of Homarus americanus (lobster), one having great affinity for mannose and fructose and low affinity for glucose.

Two major hexokinases (ATP: D-hexose 6-phosphotransferases, EC 2.7.1.1) have been identified in tissues of Homarus americanus (lobster) and separated from each other by DEAE-cellulose ion-exchange chromatography and by polyacrylamide gel electrophoresis. The molecular weight of each, determined by gel filtration, is about 50 000. Hexokinase II, named for its column elution order, resembles hexokinase isozymes I and II of vertebrates. Km values for glucose, mannose and fructose are 0.08, 0.13 and 6.7 mM, respectively. It is strongly inhibited by the reaction products, ADP and glucose-6-P (Ki = 0.8 mM). Hexokinase I appears to be different from any animal hexokinase previously described. It has a high affinity for mannose and fructose and low affinity for glucose. Km values are 6, 0.07 and 1.2 mM and relative maximum rates 100, 520 and 1070 for glucose, mannose and fructose, respectively. Hexokinase I is not inhibited by physiological concentrations of ATP nor by glucose-6-P , mannose-6-P or fructose-6-P even at high concentrations. Both enzymes occur in muscle at about 10% of the concentration found in the hepatopancreas. The use of Leuconostoc mesenteroides glucose-6-phosphate dehydrogenase (D-glucose-6-phosphate: NADP+ 1-oxidoreductase, EC 1.1.1.49), with NAD as cofactor, is recommended for measuring hexokinases in crude tissue preparations to avoid the variable further reduction of nucleotide caused by the action of 6-phosphogluconate dehydrogenase when NADP is used with yeast glucose-6-phosphate dehydrogenase.

A Limulus glucose-6-phosphatase with phosphotransferase activity characteristic of vertebrate liver microsomes. Its possible evolutionary significance.

1. Limulus hepatopancreas, coxal glands and intestine contain a particulate enzyme which can synthesize glucose 6-phosphate from glucose and inorganic pyrophosphate or carbamyl phosphate as well as hydrolyze glucose 6-phosphate. This has been clearly differentiated from hydrolysis by lysosomal or soluble phosphatases. 2. The enzyme resembles vertebrate glucose-6-phosphatase in its specific anatomical distribution, pH optimum, kinetic properties, donor specificity and phospholipid dependence, as indicated by its satency and lability to detergent treatment. 3. A variety of other invertebrates tested exhibited little or no PPi-glucose phosphotransferase activity with these properties. A similar phosphotransferase activity of lobster hepatopancreas had somewhat different kinetic properties and pH optimum. 4. The hypothesis that a specific glucose-6-phosphatase is to be found only in those animals which utilize free glucose as an important circulating form of energy is presented and discussed. It appears that a variety of transport compounds, such as trehalose and glucose, was tried at the evolutionary level of the Arthropods.