Abstract
The Galpha-interacting protein (GAIP) is known to interact with the Galphai3 protein. It has been suggested that, depending on its expression, GAIP can be a regulator of trimeric Gi protein signaling pathways. In the present study we show that the GAIP mRNA content declines during the enterocytic differentiation of two cell lines derived from human colon adenocarcinomas: HT-29 and Caco-2. In undifferentiated HT-29 cells, when the GDP/GTP cycle on the trimeric Gi3 protein is interrupted by either pertussis toxin treatment or by the transfection of a mutant of the Galphai3 protein with no GTPase activity (Q204L), we observed a decrease in the GAIP mRNA content. As these conditions are known to impair the Gi3-dependent lysosomal-autophagic pathway existing in undifferentiated HT-29 cells, we have investigated the role of GAIP in controling the lysosomal-autophagic pathway. Overexpression of GAIP stimulated protein degradation along the macroautophagic pathway. In contrast, overexpression of GAIP did not modify the low rate of macroautophagy in cells expressing the Q204L mutant of the Galphai3 protein. These results show that GAIP regulates a major catabolic pathway and that the expression of GAIP is dependent upon the activity of the Galphai3 protein and the state of enterocytic differentiation of cells.
Highlights
The best known mechanism of desensitization of trimeric G protein signaling pathways involves receptor phosphorylation [1]
In the present work we show that (i) in cells having a low capacity for macroautophagy due either to a blockade in the GDP/GTP cycle on the Gi3 protein or to their state of enterocytic differentiation the expression of G␣-interacting protein (GAIP) mRNA is reduced; (ii) overexpression of GAIP, by transfection, stimulates the lysosomal-autophagic pathway except in cells that express a mutant G␣i3 proteindeficient in GTPase activity
We have demonstrated that GAIP is a regulator of the Gi3 protein-dependent macroautophagic pathway [13, 14]
Summary
PTX, 3-MA, and all other chemicals were purchased from Sigma. Cell culture reagents and Geneticin (G418) were from Life Technologies, Inc. (Eragny, France). Enzymes for cloning and sequencing, synthetic oligonucleotides, and TrizolTM for mRNA extraction were from Life Technologies, Inc. (Les Ulis, France) and was raised against a decapeptide from the C-terminal of the protein [15]. The Rediprime random primer labeling kit, Nϩ-hybond membranes, and the radioisotopes ␣-35S-dATP (1000 Ci/mmol), [3H]raffinose (5–15 Ci/mmol), [␥-32P]GTP (Ͼ1000 Ci/mmol), L-[U-14C]valine (288.5 mCi/mmol), and 125I-labeled sheep anti-rabbit antibodies were from Amersham Corp. Reverse Transcription PCR and Cloning of GAIP cDNA were synthesized from mRNA isolated from HT-29 cells and were used to amplify a full-length cDNA encoding GAIP, using PCR. The sense PCR primer began with a BamHI site: 5Ј-CGCGGATCCATGCCCACCCCGCATGAG-3Ј. Before sequencing the PCR product was cloned into the BamHI/EcoRI sites of either the plasmid pcDNA3 (sense) or the plasmid pBK/CMV (antisense). Expression and Purification of Recombinant Proteins cDNAs encoding the G␣i3 protein or GAIP were subcloned into the His-tag fusion vector pTrcHis. cDNA inserts were downstream and in frame with a sequence that encodes a N-terminal fusion peptide con-
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