Expression cloning of an AVP-activated, calcium-mobilizing receptor from rabbit kidney medulla.
Arginine vasopressin (AVP) is a nonapeptide that regulates body fluid and blood pressure homeostasis. We have used expression cloning in the Xenopus laevis oocyte system to identify cDNA clones from a rabbit renal medullary expression library encoding an AVP receptor linked to Ca2+ mobilization. cRNA generated from positive clones conferred upon oocytes the capacity to mobilize intracellular Ca2+ in response to AVP. A cDNA clone encoding a protein of 780 amino acids was isolated, sequenced, and subcloned into an SV40-based expression vector. Expression of the cloned protein [designated the vasopressin-activated, calcium-mobilizing (VACM-1) protein] in COS-1 cells, resulted in increased 125I-labeled AVP binding [dissociation constant (Kd) of approximately 2 nM] and increased AVP-induced mobilization of Ca2+. Importantly, 125I-AVP could be immunoprecipitated both from detergent-solubilized membranes from COS-1 cells expressing VACM-1 protein and from an in vitro translation system, in which VACM-1 protein was synthesized, using antibodies prepared against a synthetic peptide derived from the NH2-terminal sequence of VACM-1. Interestingly, immunohistochemical staining of rabbit kidney sections with this antibody showed specific staining of collecting tubule epithelia. The deduced amino acid sequence is not homologous with any nucleic acid or amino acid sequences reported to date, including those of the V1 and V2 AVP receptors. The VACM-1 protein may represent a novel AVP receptor.
Burnatowska-Hledin
M A
MA
Department of Biology, Peale Science Center, Hope College, Holland 49422-9000, USA.
Spielman
W S
WS
Smith
W L
WL
Shi
P
P
Meyer
J M
JM
Dewitt
D L
DL
D000595:Amino Acid Sequence; D000818:Animals; D001127:Arginine Vasopressin / Q000494:pharmacology*; D001483:Base Sequence; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism*; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D044842:Cullin Proteins; D017931:DNA Primers; D018076:DNA, Complementary; D005260:Female; D015870:Gene Expression; D015723:Gene Library; D007668:Kidney; D007679:Kidney Medulla / Q000378:metabolism*; D008565:Membrane Proteins / Q000032:analysis / Q000096:biosynthesis*; D008969:Molecular Sequence Data; D009865:Oocytes / Q000378:metabolism; D012333:RNA, Messenger / Q000032:analysis / Q000096:biosynthesis; D011817:Rabbits; D017483:Receptors, Vasopressin / Q000032:analysis / Q000096:biosynthesis*; D011994:Recombinant Proteins / Q000032:analysis / Q000096:biosynthesis / Q000302:isolation & purification; D015183:Restriction Mapping; D014162:Transfection; D014982:Xenopus laevis
C094547:Cul5 protein, rat; D044842:Cullin Proteins; D017931:DNA Primers; D018076:DNA, Complementary; D008565:Membrane Proteins; D012333:RNA, Messenger; D017483:Receptors, Vasopressin; D011994:Recombinant Proteins; D001127:Arginine Vasopressin; D002118:Calcium