High affinity insulin binding by soluble insulin receptor extracellular domain fused to a leucine zipper.
Insulin receptors (IRs) that are truncated at the end of the ectodomain form dimers that bind insulin with different characteristics to wild type receptors. These soluble IRs have lowered affinity for insulin compared with full-length IR, and exhibit linear Scatchard plots in contrast to the curvilinear plots obtained with full-length IR, IR truncated at the C-terminus of the transmembrane region and IR ectodomains fused to the self-associating constant domains from Fc or lambda immunoglobulins. In this report, we have fused the IR ectodomain to the 33 residue leucine zipper from the transcriptional activator GCN4 of Saccharomyces cerevisiae. This fusion protein binds insulin with high affinity in a manner comparable to native receptor. The respective dissociation constants were Kd1 8.2 X 10(-11) M and Kd2 1.6 x 10(-8) M for hIRedZip and Kd1 5.7 x 10(-11) M and Kd2 6.3 x 10(-9) M for membrane-anchored, native receptor.
Hoyne
P A
PA
CSIRO, Division of Health Sciences and Nutrition, Parkville Vic, Australia.
Cosgrove
L J
LJ
McKern
N M
NM
Bentley
J D
JD
Ivancic
N
N
Elleman
T C
TC
Ward
C W
CW
D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D002460:Cell Line; D006224:Cricetinae; D017931:DNA Primers / Q000235:genetics; D019281:Dimerization; D006801:Humans; D066298:In Vitro Techniques; D007328:Insulin / Q000378:metabolism*; D007700:Kinetics; D016350:Leucine Zippers / Q000235:genetics; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D011972:Receptor, Insulin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins; D012441:Saccharomyces cerevisiae / Q000235:genetics; D012995:Solubility; D014162:Transfection
D017931:DNA Primers; D007328:Insulin; D011993:Recombinant Fusion Proteins; D011972:Receptor, Insulin