Functional characterization of male germ cell-specific CREM isoforms.
Due to alternative promoter usage, splicing, and translational initiation, expression of the cAMP-responsive element modulator (CREM) gene results in the production of functionally different CREM proteins with either activating or repressing potential on target gene expression. Recently, we demonstrated 2 novel isoforms (CREM-2-F-G-H-Ib and CREM-2-G-H-Ib) in various germ cell types during normal and impaired human spermatogenesis. In contrast to known isoforms, these exhibit a transactivation domain but lack a kinase-inducible domain (KID) domain resulting in a disruption of the open reading frame. In the present study, we functionally analyzed these isoforms. Investigation of both in vitro and in vivo expressed proteins from human testis RNA suggests that a novel upstream open reading frame in exon 2 is translated from isoform CREM-2-F-G-H-Ib, giving rise to a full-length protein. Furthermore, in both isoforms, usage of downstream adeninethymine-guanines (ATGs) for translation initiation could be observed. Sequence-specific DNA binding of CREM isoforms was confirmed by electrophoretic mobility shift assays. Luciferase reporter gene assays in cells transfected with novel CREM cDNAs demonstrated that protein kinase A dependent stimulation was inhibited by coexpression of CREM-2-F-G-H-Ib but not of CREM-2-G-H-Ib.
Jaspers
Saskia
S
Klinik für Urologie und Kinderurologie, Rudolf-Buchheim-Strasse 7, 35383 Giessen, Germany.
Gellersen
Birgit
B
Kempf
Rita
R
Samalecos
Annemarie
A
Bergmann
Martin
M
Steger
Klaus
K
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D015153:Blotting, Western; D045744:Cell Line, Tumor; D003001:Cloning, Molecular; D051786:Cyclic AMP Response Element Modulator / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D004247:DNA / Q000378:metabolism; D006801:Humans; D008297:Male; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D011485:Protein Binding; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics / Q000502:physiology; D013094:Spermatozoa / Q000502:physiology*; D014158:Transcription, Genetic
D020033:Protein Isoforms; D051786:Cyclic AMP Response Element Modulator; D004247:DNA