SECOND MESSENGERS AND CELL REGULATION 
David L. Garbers, Ph.D., Investigator 
Spermatozoan behavior is altered in various ways 
when the cells are in the vicinity of the egg. In ex- 
periments that targeted the molecular basis of signal- 
ing between the egg and the spermatozoon in the 
sea urchin, Dr. Garbers' laboratory found that one of 
the sperm receptors was the enzyme guanylyl cy- 
clase. Subsequently members of the same receptor 
family were discovered in mammalian somatic cells. 
Three particulate forms are currently known to exist 
in mammals and are designated GC-A, GC-B, and 
GC-C, in the order of the cloning of their mRNA. At 
least two other particulate forms are also known to 
exist. There are also at least four subunits of guan- 
ylyl cyclase that combine to form active hetero- 
dimers, designated al, a2, and /32. The normal 
pairing of the subunits is not known, except for 
a 1/181, where the heterodimeric protein has been 
purified from lung. During the past year, research in 
Dr. Garbers' laboratory has focused on the function 
of these guanylyl cyclase-linked receptors. 
Phenotype of the Expressed GC-C Receptor 
After the stable expression of GC-C in human em- 
bryonic kidney 293 cells, cGMP concentrations 
were elevated ~ 40-fold in response to heat-stable 
enterotoxins (STa). STa stimulated the cyclase of 
isolated membranes from these cells about ninefold. 
ATP appears to potentiate the STa stimulation, but 
unlike the situation with GC-A, the nucleotide ap- 
pears to protect GC-C against inactivation. Antibody 
prepared to the carboxyl-terminal peptide of GC-C 
immunoprecipitated two proteins of molecular 
weight 140,000 and 160,000 from the 293 cells. 
Tunicamycin treatment of the cells caused both pro- 
teins to collapse to a molecular weight of 1 20,000, 
the molecular size predicted from the cDNA se- 
quence. Therefore GC-C is an amino-linked glyco- 
protein. In preparations of rat intestinal membranes, 
three major proteins of molecular weight 65,000, 
85,000, and 140,000 were specifically recognized 
by the GC-C antibody on reducing SDS-PAGE (so- 
dium dodecyl sulfate-polyacrylamide gel electro- 
phoresis), but only a single protein of molecular 
weight 230,000 was recognized under nonreducing 
conditions. Therefore GC-C is apparently proteo- 
lyzed in the intestine but does not dissociate be- 
cause of the presence of inter- and intramolecular 
disulfide bonds. It can be concluded that many or all 
of the lower-molecular-weight STa-binding proteins 
previously reported in intestinal extracts represent 
proteolytic products of GC-C. 
Cloning and Expression of the cDNA 
for Guanylin 
A small peptide named guanylin was recently iso- 
lated from the intestine of the rat and shown to ele- 
vate cGMP and to compete with STa for receptor 
binding. In studies designed to determine whether 
guanylin represents the endogenous ligand for GC- 
C, the cDNA encoding this peptide was cloned. The 
mRNA encodes an open reading frame of 1 1 5 amino 
acids; the 1 5-residue guanylin was found at the car- 
boxyl terminus. The peptide initially isolated likely 
represents an isolation artifact, however, because an 
acid-labile Asp/Pro bond exists at the amino ter- 
minus. Transfection of COS-7 cells with the guany- 
lin cDNA yielded a secreted protein with a mo- 
lecular weight of ~ 10,000, but the expressed 
proguanylin failed to stimulate GC-C unless it was 
cleaved to yield lower-molecular-weight peptides. 
Guanylin mRNA was detected in the intestine, kid- 
ney, adrenal gland, and uterus/oviduct of the rat by 
Northern hybridization. GC-C, the apparent recep- 
tor for guanylin, was detected in the adrenal gland, 
airway epithelial cells, brain, and olfactory and tra- 
cheal mucosa by Northern analysis, the polymerase 
chain reaction, or cDNA cloning. Therefore guany- 
lin and its putative receptor exist in various mam- 
malian tissues. 
Mechanisms of Guanylyl Cyclase 
Desensitization 
When GC-A, the receptor for atrial natriuretic 
peptide (ANP), was stably expressed in human em- 
bryonic kidney 293 cells, cellular cGMP concentra- 
tions were transiently elevated by ANP. GC-A was 
subsequently shown to exist as a phosphoprotein 
under basal conditions, and the addition of ANP 
caused a decrease in ^^P content. Coincident with 
the loss of '^P, GC-A showed a marked reduction in 
ANP-stimulable activity. The addition of protein 
phosphatase 2A also caused both the disappearance 
of ^^P from GC-A and a loss of responsiveness to 
ANP, suggesting that GC-A is desensitized by de- 
phosphorylation . 
Mechanisms of Signal Transduction 
by Guanylyl Cyclases 
Transduction of the binding signal in growth fac- 
tor receptors appears to be associated intimately 
with a ligand-induced aggregation of receptors. The 
guanylyl cyclase receptors are similar to growth fac- 
tor receptors in the sense they are about the same 
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