5 OUT OF 6 TRYPTOPHAN RESIDUES IN THE N-TERMINAL EXTRACELLULAR DOMAINOF THE RAT GLP-1 RECEPTOR ARE ESSENTIAL FOR ITS ABILITY TO BIND GLP-1

Oligonucleotide-directed mutagenesis was utilized to investigate the r equirement of tryptophan residues located in the N-terminal domain of the glucagon-like peptide-1 (GLP-1) receptor for the ability to bind i ts ligand and to induce cAMP generation. W-39, W-72, W-87, W-91, W-110 , and W-120 were mutated into alanine. Two of the six tryptophan resid ues, W-72 and W-110, are highly conserved within the receptor subfamil y. After transfection of mutated cDNAs in COS-7 or CHL cells, it appea red that mutant W(87)A bound [I-125] GLP-1 with the same affinity as w ild-type receptor and induced signal transduction to a comparable exte nt. In contrast, mutant receptors W(39)A, W(72)A, W(91)A, W(110)A, and W(120)A lost the ability to bind [I-125]GLP-1. Because all mutated re ceptor cDNAs were transcribed on RNA level (Northern blot) and the rec eptor proteins were expressed at the plasma membrane level (Western bl ot), it is concluded that with the exception of W-87 all tryptophan re sidues are essential for receptor ligand interaction. This indicates t he significance of hydrophobic interactions within the N-terminal doma in of the GLP-1 receptor. (C) 1997 Elsevier Science Inc.