Characterization of the carboxyl-terminal domain of the rat glucose-dependent insulinotropic polypeptide (GIP) receptor. A role for serines 426 and 427 in regulating the rate of internalization.
Glucose-dependent insulinotropic polypeptide (GIP) is a gastrointestinal hormone involved in the regulation of insulin secretion. In non-insulin-dependent diabetes mellitus insulin responses to GIP are blunted, possibly due to altered signal transduction or reduced receptor number. Site-directed mutagenesis was used to construct truncated GIP receptors to study the importance of the carboxyl-terminal tail (CT) in binding, signaling, and receptor internalization. Receptors truncated at amino acids 425, 418, and 405, expressed in COS-7 or CHO-K1 cells, exhibited similar binding to wild type receptors. GIP-dependent cAMP production with the 405 mutant was decreased in COS-7 cells. Maximal cAMP production in CHO-K1 cells was reduced with all truncated forms. Binding was undetectable with a receptor truncated at amino acid 400; increasing tail length by adding 5 alanines restored binding and signaling. Mutants produced by alanine scanning of residues 394-401, adjacent to transmembrane domain 7, were all functional. CT truncation by 30 or more amino acids, mutation of serines 426/427, singly or combined, or complete CT serine knockout all reduced receptor internalization rate. The majority of the GIP receptor CT is therefore not required for signaling, a minimum chain length of approximately 405 amino acids is needed for receptor expression, and serines 426 and 427 are important for regulating rate of receptor internalization.
Wheeler
M B
MB
Departments of Medicine and Physiology, University of Toronto, Toronto, Ontario M5S 1A8, Canada.
Gelling
R W
RW
Hinke
S A
SA
Tu
B
B
Pederson
R A
RA
Lynn
F
F
Ehses
J
J
McIntosh
C H
CH
D000818:Animals; D016466:CHO Cells; D019556:COS Cells; D006224:Cricetinae; D000242:Cyclic AMP / Q000378:metabolism; D005947:Glucose / Q000494:pharmacology; D007328:Insulin / Q000378:metabolism; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D051381:Rats; D011964:Receptors, Gastrointestinal Hormone / Q000737:chemistry* / Q000235:genetics; D017384:Sequence Deletion; D015398:Signal Transduction; D014162:Transfection
D007328:Insulin; D011964:Receptors, Gastrointestinal Hormone; C041086:gastric inhibitory polypeptide receptor; D000242:Cyclic AMP; D005947:Glucose