Cloning of the mRNA for the protein that crosslinks to the egg peptide speract.
An apparent receptor for the egg peptide speract (Gly-Phe-Asp-Leu-Asn-Gly-Gly-Gly-Val-Gly) was identified by covalently coupling a radiolabeled speract analogue to intact spermatozoa and was then purified by DEAE-Sepharose chromatography and preparative gel electrophoresis after solubilization with Lubrol PX. The purified, crosslinked protein was digested with Staphylococcus aureus V8 protease and a resultant peptide, purified from polyacrylamide slab gel slices, was shown to have the amino acid sequence Val-Ser-Ala-Pro-Phe-Asp-Leu-Glu-Ala-Pro-Phe-Ile-Ile-Asp-Gly-Ile. Polyclonal antiserum, generated against a synthetic peptide that corresponded to the above sequence, immunoprecipitated the radiolabeled crosslinked protein and reacted with a Mr 77,000 protein on immunoblots, demonstrating that the sequenced peptide originated from the apparent receptor. A clone containing a 2.5-kilobase insert was subsequently isolated from a sea urchin testis cDNA library that contained DNA sequences encoding an open reading frame of 532 amino acids that included the above peptide sequence. The deduced amino acid sequence suggests that the protein contains a 26-residue amino-terminal signal peptide, a large extracellular domain relatively rich in cysteine (5%) that includes a four-fold repeat of about 115 amino acids, a single membrane-spanning region, and only 12 amino acid residues extending into the cytoplasm. Analysis of total RNA from Strongylocentrotus purpuratus testis by Northern blot revealed a 2.5-kilobase RNA. Preliminary data show the presence of hybridizing RNA of the same apparent size in other sea urchin species, including Arbacia punctulata, which does not respond to speract.
Dangott
L J
LJ
Howard Hughes Medical Institute, Vanderbilt University Medical Center, Nashville, TN 37232.
Jordan
J E
JE
Bellet
R A
RA
Garbers
D L
DL
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D003432:Cross-Linking Reagents; D005260:Female; D008297:Male; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009842:Oligopeptides / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics*; D011956:Receptors, Cell Surface / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D012091:Repetitive Sequences, Nucleic Acid; D015183:Restriction Mapping; D012617:Sea Urchins; D013084:Sperm-Ovum Interactions
D003432:Cross-Linking Reagents; D009842:Oligopeptides; D012333:RNA, Messenger; D011956:Receptors, Cell Surface; C035027:egg surface sperm receptor; C028364:speract