Glucose-dependent insulinotropic polypeptide (GIP) is an incretin hormone involved in regulating glucose and lipid metabolism. GIP receptor (GIPR) antagonism is believed to offer therapeutic ...potential for various metabolic diseases. Pharmacological intervention of GIPR, however, has limited success due to lack of effective antagonistic reagents. Previously we reported the discovery of two mouse anti-murine GIPR monoclonal antibodies (mAbs) with distinctive properties in rodent models. Here, we report the detailed structural and biochemical characterization of these two antibodies, mAb1 and mAb2. In vitro and in vivo characterizations demonstrated mAb2 is a full GIPR antagonistic antibody and mAb1 is a non-neutralizing GIPR binder. To understand the molecular basis of these two antibodies, we determined the co-crystal structures of GIPR extracellular domain in complex with mAb1 and with mAb2 at resolutions of 2.1 and 2.6 Å, respectively. While the non-neutralizing mAb1 binds to GIPR without competing with the ligand peptide, mAb2 not only partially occludes the ligand peptide binding, but also recognizes the GIPR C-terminal stalk region in a helical conformation that acts as a molecular mimic of the ligand peptide and locks GIPR in a novel auto-inhibited state. Furthermore, administration of mAb2 in diet-induced obesity mice for 7 weeks leads to both reduction in body weight gain and improvement of metabolic profiles. In contrast, mAb1 has no effect on body weight or other metabolic improvement. Together, our studies reveal the unique molecular mechanism of action underlying the superior antagonistic activity of mAb2 and signify the promising therapeutic potential of effective GIPR antagonism for the treatment of metabolic disorders.
Homogeneous staining region (hsr), a cytogenetic indicator of gene amplification, has been frequently found in ovarian carcinoma (ovc). To identify the origin of the hsr, chromosome microdissection ...combined with polymerase chain reaction and fluorescence in situ hybridization (FISH) was applied to two human ovarian cancer cell lines, GR and MLS/P. The hsr probes were labeled with biotin or digoxigenin and hybridized to normal metaphase spreads to elucidate the chromosomal origin and regional localization of the amplified genes. FISH to normal metaphase spreads with the probe generated from the whole hsr-bearing chromosome from GR hybridized to 8q24, 2p13
→2q11.2, 10pter
→10p15, 10p12
→10q11.2, 5q23
→5q31, and 5q33
→5qter. For MLS/P, the hsr-bearing marker chromosome hybridized to 8q and 15q. In both cases, detailed FISH analysis revealed enhanced signal intensity at the 8q24 locus, which coincides with the chromosomal location of the C-
MYC oncogene. To verify the involvement of C-
MYC in hsr formation, in situ hybridization with a probe specific for the C-
MYC oncogene was conducted and confirmed the amplification of C-
MYC as the origin of the hsr. The whole hsr-bearing chromosome for GR is designated as rev ish der(10) (10pter
→10p15::8q24hsr:: 10p12
→10q11.2::8q24::2q11.2
→2p13::2p13
→2q11.2::8q24::10q11
→10p11.2::5q23
→5q31::5q33
→5qter (wcp10
+,D10Z1
++,wcp2
+,D2Z
++,wcp5
+,wcp8
+,C-MYC
++/hsr). The hsr-bearing marker for MLS/P is designated as rev ish der(8)(qter
→8q24::8q24::8q24
→8q10::8q10
→8q24::8q24::8q24::8q24
→8qter:: 15q11
→15qter)(wcp8
+,D8Z1
+,wcp15
+,C-
MYC+++). FISH with the probe generated from the hsr of GR also painted the hsr in MLS/P, indicating that the two hsrs have shared homology, which indicates that the amplification of 8q24/C-
MYC as the origin of hsr may be a nonrandom genomic alteration in ovc.
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