FIGURE 1.

Identification of a cGMP-response element-binding protein. A, yeast one-hybrid screening of a human kidney cDNA library; the putative binding protein interacts with cGMP-RE (present in three copies) to activate the reporter gene via GAL4AD. B, full-length mRNA determination was by primer extension. HEK293 total RNA was retrotranscribed using a radiolabeled primer targeting GREBP mRNA and run beside pACT2-GREBP plasmid sequencing with the same radioactive primer. C, full-length GREBP mRNA was confirmed by RT-PCR with a specific primer targeting upstream and downstream regions of the transcription start site. The positive control was HEK293 DNA, whereas retrotranscribed HEK293 RNA was studied by −25/−5 and +1/+20 RT-PCR. D, nucleotide and amino acid sequences of GREBP. The mRNA destabilization motif is double underlined. Basic residues (framed) constitute 20% of GREBP (23 of 115) and are distributed along the whole sequence. Boldface residues are those with high possibility of serine phosphorylation (>0.800:1.00), and threonine could be strongly phosphorylated (>0.900:1.00) by serine/threonine kinase, including PKG.