MOLECULAR BASIS OF FERTILIZATION 
David L. Garbers, Ph.D., Investigator 
The major focus of this research continues to be 
the molecular basis of fertilization, with emphasis 
on the structures of those molecules that specific- 
ally interact with spermatozoan receptors, the iden- 
tification of the receptor molecules, and the defini- 
tion of the physiological events that occur in 
response to receptor occupation. The results ob- 
tained with germ cells also provide important infor- 
mation on the mechanisms by which other cells re- 
spond to their environment. 
I. Primary Structure of the Membrane Form of 
Guanylate Cyclase. 
Initially the mRNA encoding guanylate cyclase 
from the sea urchin Arbacia punctulata was 
cloned. Crosslinking studies in Arbacia had sug- 
gested that guanylate cyclase was the receptor for 
an egg-derived peptide. The cDNA from this sea ur- 
chin was used to probe for hybridizing clones in 
cDNA libraries from another species of sea urchin, 
as well as from rat and human tissues. 
A cDNA clone for the membrane form of guanyl- 
ate cyclase was isolated from the testis of the sea 
urchin Strongylocentrotus purpuratus. An open 
reading frame predicted a protein of 1,125 amino 
acids including an apparent signal peptide of 21 
residues. The deduced protein sequence was ho- 
mologous to the protein kinase family and con- 
tained limited but significant regions of identity 
with a low-molecular-weight atrial natriuretic pep- 
tide-clearance (ANP-C) receptor. The carboxyl re- 
gion (202 amino acids) was 42% identical with a 
subunit of the cytoplasmic form of guanylate cy- 
clase cloned from bovine lung but was distinctly 
different from the Arbacia sequence in the distal 
carboxyl region. 
The sea urchin clone was successfully used to 
isolate positive-hybridizing clones from rat brain 
and human kidney and placental cDNA libraries. 
The deduced sequences of the human and rat 
clones were nearly identical. The open reading 
frame of the rat brain cDNA encoded a protein of 
1,057 amino acids, including a predicted 28-amino 
acid signal peptide. 
Further analysis of the deduced amino acid se- 
quence of the guanylate cyclase cDNA indicated 
that it could be divided into three potential func- 
tional domains, based on homology with other pro- 
teins. The extracellular domain of the rat mem- 
brane guanylate cyclase is 33% identical with the 
bovine ANP-C receptor, an ANP-binding protein that 
is apparently not coupled to activation of guanylate 
cyclase. The ANP-C receptor consists of an extracel- 
lular ANP-binding domain, a transmembrane do- 
main, and a short (37 amino acids) cytoplasmic tail. 
The five cysteine residues of the ANP-C receptor are 
conserved in guanylate cyclase. 
Just within the transmembrane domain an intra- 
cellular domain related to the catalytic domain of 
protein kinases is found, although protein kinase 
activity has not yet been detected. A 256-amino 
acid portion of the intracellular domain is 31% 
identical to the protein tyrosine kinase domain of 
the platelet-derived growth factor receptor. Guanyl- 
ate cyclase conforms to the protein kinase consen- 
sus sequence in 30 of 33 residues highly conserved 
or invariant across the protein kinase family. The 
Gly-X-Gly-X-X-Gly consensus sequence of protein ki- 
nases, however, is Gly-X-Gly-X-X-X-Gly in the rat 
guanylate cyclase. 
The highest degree of similarity between the de- 
duced amino acid sequence of the membrane form 
of guanylate cyclase and other proteins is found in 
the carboxyl portion of the intracellular domain. A 
253-amino acid sequence in this region is 42% 
identical to the carboxyl terminus of one of the 
subunits of a bovine soluble form of guanylate cy- 
clase. It is not known whether the sequence deter- 
mined for the soluble enzyme is from a regulatory 
or catalytic subunit. In addition, the bovine brain 
adenylate cyclase contains the two internally ho- 
mologous domains that are also homologous to 
this region of guanylate cyclase. 
II. Guanylate Cyclase as a Cell-Surface Receptor. 
The cloned guanylate cyclase functions as an ANP 
receptor. Cells transfected with a vector containing 
the rat brain clone specifically bind ~9 times more 
^^^I-labeled ANP than cells transfected with vector 
alone. ^^^I-ANP binds with high affinity and the ex- 
pected specificity (half-maximal inhibition of bind- 
ing of 0.4 nM ^^^I-ANP occurred at ~3 nM unla- 
beled ANP or 100 nM unlabeled atriopeptin I). To 
verify that guanylate cyclase acts as an ANP recep- 
tor, ^^^I-ANP was used in crosslinking experiments. 
Transfected cells were incubated with ^^^I-ANP, 
with or without an excess of unlabeled ANP After 
crosslinking and SDS-PAGE (reducing conditions), a 
Continued 
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