TY - JOUR
T1 - Functional analysis of glucose-dependent insulinotropic polypeptide fusion proteins
AU - Ding, Ke Hong
AU - Isales, Carlos M.
AU - Zhong, Qing
AU - Bollag, Roni J.
N1 - Funding Information:
The authors thank Dr. Li Zhong for his assistance with Western blot analysis. This work was supported by NIH grant HD34149 (RJB) and a Medical College of Georgia Research Institute grant (CMI).
PY - 2001
Y1 - 2001
N2 - 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 Ca2+ 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.
AB - 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 Ca2+ 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.
KW - Fusion protein
KW - Glucose-dependent insulinotropic polypeptide (GIP)
KW - Green fluorescent protein (GFP)
KW - Peptide secretion
KW - Signal transduction
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U2 - 10.1016/S0196-9781(01)00366-7
DO - 10.1016/S0196-9781(01)00366-7
M3 - Article
C2 - 11311726
AN - SCOPUS:0035043770
SN - 0196-9781
VL - 22
SP - 575
EP - 582
JO - Peptides
JF - Peptides
IS - 4
ER -