Abstract
Guanylyl cyclase C (GC-C) is a newly discovered receptor found in the intestine, and possibly in other epithelia, that binds bacterial heat-stable enterotoxins (STa). The receptor has now been stably expressed in human embryonic 293 cells, which do not normally contain the receptor. Cyclic GMP concentrations are elevated 40-fold in response to 1 microM STa, and membranes obtained from the overproducing cells contain GC-C activity that can be stimulated about 9-fold by STa alone and an additional 1.4- to 2-fold by a combination of ATP and STa. The ATP effect does not appear to be due to enzyme activation, but instead to protection of GC-C against inactivation. Antibody raised against the carboxyl-terminal sequence of GC-C identified two major proteins (M(r) 140,000 and 160,000) in 293 cells expressing GC-C. Both proteins bound to wheat germ lectin-Sepharose, and N-glycosidase F treatment converted both forms to a single M(r) 120,000 protein, the size predicted from amino acid composition. The addition of high concentrations of tunicamycin to 293-GC-C cells also reduced the M(r) to 120,000, indicating that GC-C is an N-linked glycoprotein. When rat intestinal membranes or 293-GC-C cells were cross-linked with 125I-labeled STa, the major 125I-labeled protein complexes had M(r) ranging between 45,000 and 80,000. On immunoblots of rat intestinal membranes treated with a reducing agent, 3 major proteins of M(r) 65,000, 85,000, and 140,000 were specifically recognized by a GC-C antibody. However, under nonreducing conditions one major GC-C related protein appeared at a higher position (M(r) 230,000). Its mobility was reduced in the presence of STa, similar to rCG-C expressed in 293 cells. These data indicate that at least part of the lower M(r) STa-binding proteins found in intestinal extracts represent proteolytic products of GC-C.
Highlights
Guanylylcyclase C (GC-C) is a newlydiscovered activate signal transduction pathwaysnormally exploited by receptor found in the intestine, and possibly in other endogenous neurohormonal regulators of intestinal salt and epithelia, that binds bacterial heat-stable enterotoxinws ater transport [2]
When r a t intestinal subsequent expression of these clones in mammalian cells, membranes or 293-GC-C cells were cross-linked with revealed that an intestinal guanylyl cyclase (GC-C), ‘261-labeledSTa, the major ‘2SI-labeledprotein com- was a stable enterotoxins (STa) receptor [9,10,11]
T o investigate the phenotypeof the cloned rat STa recepincubation for 30 min a t 37 "C, membranes were washed, resuspended tor/guanylyl cyclase,we stably expressed the protein i n 50 pl of SDS samplebuffer, and analyzedby SDS-PAGE followed in human embryonickidney 293 cells
Summary
Were resuspended 100p l of DMEM, pH7.4, and 1mM disuccinimidyl suberate in dimethylsulfoxide was added froma 100 mM stock. T o investigate the phenotypeof the cloned rat STa recepincubation for 30 min a t 37 "C, membranes were washed, resuspended tor/guanylyl cyclase (rGC-C),we stably expressed the protein i n 50 pl of SDS samplebuffer, and analyzedby SDS-PAGE followed in human embryonickidney 293 cells. Guanylyl Cyclase Assay and Cyclic GMP Determinations-Membranes (25-50 pg of protein) were incubated in a final volume of 0.1 ml containing 100 mM NaC1, 50 mM Tris/HClpH 7.4, 0.25 mM IBMX, 10 mM MgC12,5 mM creatine phosphate, 3-5 units of creatine phosphokinase, and 1 mM GTPat 37 "C. The cGMP rGC-C cells from 1.5 & 0.3 to 68 f 25 pmol/mg protein ( n= 3)during a 10-minincubationinthe presence of 0.5 mM IBMX.The guanylyl cyclase (GC) of isolated293-rGC-C membranes could be stimulated approximately9-fold by STa, the cGMPproduced being 31 1 8 pmol of cGMP/10 min/mg was determined in the HC1-containing fraction after centrifugation of protein under basal conditions a2n9d5 f 43 pmolof cGMP/.
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