Structural determinants for activity of glucagon-like peptide-2.
Glucagon-like peptide-2 (GLP-2) is a 33 amino acid gastrointestinal hormone that regulates epithelial growth in the intestine. Dipeptidylpeptidase IV cleaves GLP-2 at the position 2 alanine, resulting in the inactivation of peptide activity. To understand the structural basis for GLP-2 action, we studied receptor binding and activation for 56 GLP-2 analogues with either position 2 substitutions or alanine replacements along the length of the peptide. The majority of position 2 substitutions exhibited normal to enhanced GLP-2 receptor (GLP-2R) binding; in contrast, position 2 substitutions were less well tolerated in studies of receptor activation as only Gly, Ile, Pro, alpha-aminobutyric acid, D-Ala, or nor-Val substitutions exhibited enhanced GLP-2R activation. In contrast, alanine replacement at positions 5,6,17, 20, 22, 23, 25, 26, 30, and 31 led to diminished GLP-2R binding. Position 2 substitutions containing Asp, Leu, Lys, Met, Phe, Trp, and Tyr, and Ala substitutions at positions 12 and 21 exhibited normal to enhanced GLP-2R binding but greater than 75% reduction in receptor activation. D-Ala(2), Pro(2) and Gly(2), Ala(16) exhibited significantly lower EC(50)s for receptor activation than the parent peptide (p < 0.01-0.001). Circular dichroism analysis indicated that the enhanced activity of these GLP-2 analogues was independent of the alpha-helical content of the peptide. These results indicate that single amino acid substitutions within GLP-2 can confer structural changes to the ligand-receptor interface, allowing the identification of residues important for GLP-2R binding and receptor activation.
Departments of Physiology, University of Toronto, Toronto, Canada, M5S 1A8.
M P
MP
DaCambra
B
B
Yusta
M
M
Sumner-Smith
A
A
Crivici
D J
DJ
Drucker
P L
PL
Brubaker
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D002460:Cell Line; D002942:Circular Dichroism; D053767:Glucagon-Like Peptide 2; D000067757:Glucagon-Like Peptide-1 Receptor; D004763:Glucagon-Like Peptides; D006801:Humans; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000235:genetics* / Q000378:metabolism*; D051381:Rats; D018027:Receptors, Glucagon / Q000378:metabolism / Q000502:physiology; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship
C000593833:GLP1R protein, human; C000593835:Glp1r protein, rat; D053767:Glucagon-Like Peptide 2; D000067757:Glucagon-Like Peptide-1 Receptor; D010446:Peptide Fragments; D018027:Receptors, Glucagon; D004763:Glucagon-Like Peptides