Characterization of the thyroid Na+/I- symporter with an anti-COOH terminus antibody.
The Na+/I- symporter (NIS) is the plasma membrane protein that catalyzes active I- transport in the thyroid, the first step in thyroid hormone biogenesis. The cDNA encoding NIS was recently cloned in our laboratory and a secondary structure model proposed, suggesting that NIS is an intrinsic membrane protein (618 amino acids; approximately 65.2 kDa predicted molecular mass) with 12 putative transmembrane domains. Here we report the generation of a site-directed polyclonal anti-COOH terminus NIS antibody (Ab) that immunoreacts with a approximately 87 kDa-polypeptide present in membrane fractions from a rat thyroid cell line (FRTL-5). The model-predicted cytosolic-side location of the COOH terminus was confirmed by indirect immunofluorescence experiments using anti-COOH terminus NIS Ab in permeabilized FRTL-5 cells. Immunoreactivity was competitively blocked by the presence of excess synthetic peptide. Treatment of membrane fractions from FRTL-5 cells, Xenopus laevis oocytes, and COS cells expressing NIS with peptidyl N-glycanase F converted the approximately 87 kDa-polypeptide into a approximately 50 kDa-species, the same relative molecular weight exhibited by NIS expressed in E. coli. Anti-NIS Ab immunoprecipitated both the NIS precursor molecule (approximately 56 kDa) and the mature approximately 87 kDa form. Furthermore, a direct correlation between circulating levels of thyroid-stimulating hormone and NIS expression in vivo was demonstrated.
Levy
O
O
Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, NY 10461, USA.
Dai
G
G
Riedel
C
C
Ginter
C S
CS
Paul
E M
EM
Lebowitz
A N
AN
Carrasco
N
N
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies; D019556:COS Cells; D002352:Carrier Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D019084:Fluorescent Antibody Technique, Indirect; D008565:Membrane Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D008961:Models, Structural; D008969:Molecular Sequence Data; D009865:Oocytes / Q000502:physiology; D017433:Protein Structure, Secondary; D011817:Rabbits; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D027981:Symporters; D014162:Transfection; D014982:Xenopus laevis; D000099209:Sodium-Iodide Symporters
D000906:Antibodies; D002352:Carrier Proteins; D008565:Membrane Proteins; D011994:Recombinant Proteins; D027981:Symporters; D000099209:Sodium-Iodide Symporters