Changes in the abundance of androgen receptor isotypes: effects of ligand treatment, glutamine-stretch variation, and mutation of putative phosphorylation sites.
The SDS-polyacrylamide gel electrophoresis (SDS-PAGE) migration pattern of wild-type and mutated human androgen receptors (ARs) expressed in COS-1 cells was analyzed. In the absence of hormone, the wild-type AR migrated as a closely spaced 110-112 kDa doublet. Alkaline phosphatase treatment resulted in a single 110 kDa band showing that the 112 kDa upshift reflects receptors phosphorylation. Deletion of the N-terminal amino acids 46-101 or 100-142 resulted in mutant ARs migrating as single protein bands. Three consensus phosphorylation sites in this region were substituted, and the resulting mutated proteins were analyzed. Two Ser-Pro-directed kinase consensus sites at positions Ser-80 and Ser-93 were both necessary for the AR 112 kDa upshift. Substitution of the putative casein kinase II Ser-118 site had no effect on the AR migration pattern. Surprisingly, deletion of the glutamine repeat, located directly N-terminal of the Ser-Pro sites, resulted also in an AR single form. Lengthening of the glutamine repeat caused an increase in the spacing between the two isotypes of the doublet, showing that the number of glutamine residues determines the extent of the upshift. Hormone treatment induced an extra isotype with an apparent molecular mass of 114 kDa, resulting in a 110-112-114 kDa AR triplet. The hormone-induced upshift was dependent on the Ser-80 consensus phosphorylation site. Mutations in the DNA binding domain caused a different distribution of receptor protein over the three AR isotypes.(ABSTRACT TRUNCATED AT 250 WORDS)
Jenster
G
G
Department of Endocrinology & Reproduction, Erasmus University Rotterdam, The Netherlands.
de Ruiter
P E
PE
van der Korput
H A
HA
Kuiper
G G
GG
Trapman
J
J
Brinkmann
A O
AO
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D003062:Codon; D017353:Gene Deletion; D005973:Glutamine / Q000737:chemistry*; D006801:Humans; D015151:Immunoblotting; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D016133:Polymerase Chain Reaction; D011944:Receptors, Androgen / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D012694:Serine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013329:Structure-Activity Relationship; D014158:Transcription, Genetic
D003062:Codon; D011944:Receptors, Androgen; D005973:Glutamine; D012694:Serine