Functional analysis of glucose-dependent insulinotropic polypeptide fusion proteins.
To generate functional fluorescently tagged glucose-dependent insulinotropic polypeptide (GIP), a series of GIP expression constructs were devised. These included G1 (complete preprohormone), G2 (lacking the C-terminal extension), G3 (lacking both N- and C-terminal extensions), G4 (G2 fused to green fluorescent protein, GFP), and G5 (G3 fused to GFP). Expression of G5 in bacteria generated immunopositive GIP together with GFP fluorescence, while G4 generated only fluorescence without immunoreactivity. Transfection of NIH3T3 cells with cDNAs of G1, G3, G5, but not G2, G4, and EGFP, resulted in immunologically detectable GIP formation, although fluorescence could be detected in the latter two. GIP as well as GIP-GFP secreted by NIH3T3 cells significantly stimulated intracellular cAMP accumulation and Ca(2+) mobilization in SaOS2 cells. The GIP receptor antagonist GIP(7-30) abolished these responses. These results suggest that a GIP-GFP fusion protein seven times larger than the native peptide retains function and may be used as an in vivo probe to detect GIP receptor distribution and to explore GIP's biological roles.
Ding
K H
KH
Institute of Molecular Medicine and Genetics, Medical College of Georgia, Augusta, Georgia 30912, USA.
Isales
C M
CM
Zhong
Q
Q
Bollag
R J
RJ
D016475:3T3 Cells; D000818:Animals; D002118:Calcium / Q000378:metabolism; D017931:DNA Primers; D018076:DNA, Complementary; D005749:Gastric Inhibitory Polypeptide / Q000378:metabolism*; D049452:Green Fluorescent Proteins; D006801:Humans; D008164:Luminescent Proteins / Q000378:metabolism; D051379:Mice; D014407:Tumor Cells, Cultured
D017931:DNA Primers; D018076:DNA, Complementary; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins; D005749:Gastric Inhibitory Polypeptide; D002118:Calcium