Modulation of the receptor binding affinity of amphiregulin by modification of its carboxyl terminal tail.
Amphiregulin (AR), a heparin-binding, epidermal growth factor (EGF) receptor ligand has homology with EGF but exhibits a lower affinity for the EGF receptor than EGF. As the mature form of AR is truncated at the C terminus and lacks a conserved leucine residue known to be essential for high affinity binding of EGF to the EGF receptor, wild-type AR (AR1-84), a C-terminally extended AR construct incorporating six residues from the predicted coding sequence of AR (AR1-90) and a similarly extended construct with a Met86 to Leu substitution (AR1-90(leu86)) were expressed as recombinant proteins in yeast, purified by heparin affinity and C18 reverse phase chromatography and their relative biological activities determined. The growth factors were tested in mitogenesis and EGF receptor autophosphorylation assays and their relative order of potencies was found to be leu86 > met86 > wt. The AR1-90(leu86) construct was found to be 50- to 100-fold more active than wild type AR1-84 consistent with previously reported studies of the role of the equivalent C-terminal leucine in EGF or TGF alpha. Significantly, the C-terminally extended form of AR, AR1-90, which utilized six residues from the predicted coding sequence, was 10-times more active than wild type AR1-84. This difference in activity of the C-terminally extended form of AR may be of biological significance since differential proteolytic processing of the AR precursor in vivo could result in production of multiple forms of the growth factor with differing affinities for the EGF receptor and hence differing biological potencies.
Adam
R
R
CRC Wessex Regional Medical Oncology Unit, Southampton General Hospital, UK.
Drummond
D R
DR
Solic
N
N
Holt
S J
SJ
Sharma
R P
RP
Chamberlin
S G
SG
Davies
D E
DE
D066258:Amphiregulin; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D066255:EGF Family of Proteins; D004815:Epidermal Growth Factor / Q000378:metabolism; D066246:ErbB Receptors / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D006023:Glycoproteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D006133:Growth Substances / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins; D051379:Mice; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011994:Recombinant Proteins / Q000096:biosynthesis; D014162:Transfection
C587848:AREG protein, human; D066258:Amphiregulin; C587849:Areg protein, mouse; D066255:EGF Family of Proteins; D006023:Glycoproteins; D006133:Growth Substances; D036341:Intercellular Signaling Peptides and Proteins; D011994:Recombinant Proteins; D004815:Epidermal Growth Factor; D066246:ErbB Receptors