Inhibitor-1 (I-1) is a selective inhibitor of protein phosphatase-1 (PP1) and 
regulates several PP1-dependent signaling pathways, including cardiac 
contractility and regulation of learning and memory. The human I-1 gene has been 
spliced to generate two alternative mRNAs, termed I-1alpha and I-1beta, encoding 
polypeptides that differ from I-1 in their C-terminal sequences. Reverse 
transcription-PCR established that I-1alpha and I-1beta mRNAs are expressed in a 
developmental and tissue-specific manner. Functional analysis of I-1 in a 
Saccharomyces cerevisiae strain dependent on human I-1 for viability established 
that a novel domain encompassing amino acids 77-110 is necessary for PP1 
inhibition in yeast. Expression of human I-1 in S. cerevisiae with a partial 
loss-of-function eukaryotic initiation factor-2alpha (eIF2alpha) kinase (Gcn2p) 
mutation permitted growth during amino acid starvation, consistent with the 
inhibition of Glc7p/PP1, the yeast eIF2alpha phosphatase. In contrast, human 
I-1alpha, which lacks amino acids 83-134, and I-1 with C-terminal deletions were 
significantly less effective in promoting yeast growth under starvation 
conditions. These data suggest that C-terminal sequences of I-1 enhance 
regulation of the eukaryotic eIF2alpha phosphatase. In vitro studies established 
that C-terminal sequences, deleted in both I-1alpha and I-1beta, enhance PP1 
binding and inhibition. Expression of full-length and C-terminally truncated I-1 
in HEK293T cells established the importance of the I-1 C terminus in transducing 
cAMP signals that promote eIF2alpha phosphorylation. This study demonstrates 
that multiple domains in I-1 target cellular PP1 complexes and establishes I-1 
as a cellular regulator of eIF2alpha phosphorylation.