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

Michael B Wheeler,Richard W Gelling,Simon A Hinke,Ba Tu,Raymond A Pederson,Francis Lynn,Jan Ehses,Christopher H.S Mcintosh

doi:10.1074/jbc.274.35.24593

Michael B Wheeler, Richard W Gelling + Show 6 more

Open Access

https://doi.org/10.1074/jbc.274.35.24593

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Abstract

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.

Highlights

Glucose-dependent insulinotropic polypeptide (GIP) is a gastrointestinal hormone involved in the regulation of insulin secretion
It is concluded that the majority of the GIP receptor carboxyl-terminal tail (CT) is not required for signaling, a minimum length of the tail, rather than the specific constituent amino acids, is important for transport and insertion of the receptor into the plasma membrane, and that Ser-426 and Ser-427 are involved in receptor internalization
Receptors truncated at residues 405, 418, and 425 exhibited high affinity binding in competition binding experiments, similar to that seen in cells expressing the 455-amino acid wild type receptor (GIP-R-455) in both transient studies with COS-7 cells (Figs. 1 and 2A) and in stable cell lines generated in CHO-K1 cells (Fig. 2B, Table I)

Summary

A ROLE FOR SERINES 426 AND 427 IN REGULATING THE RATE OF INTERNALIZATION*

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. Carboxyl-terminally truncated mutants of the receptor, produced by sitedirected mutagenesis and expressed in COS-7 and CHO-K1 cells, were studied with a view to determining the effect of such mutations on ligand binding, stimulation of G-protein-coupling to adenylyl cyclase, and ligand-induced internalization. It is concluded that the majority of the GIP receptor CT is not required for signaling, a minimum length of the tail, rather than the specific constituent amino acids, is important for transport and insertion of the receptor into the plasma membrane, and that Ser-426 and Ser-427 are involved in receptor internalization

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION

89 Ϯ 41 70 Ϯ 16 106 Ϯ 39 89 Ϯ 45 80 Ϯ 31 73 Ϯ 10 116 Ϯ 7 164 Ϯ 16