Role of the glucagon receptor COOH-terminal domain in glucagon-mediated signaling and receptor internalization.
The binding of glucagon to its hepatic receptor is known to result in a number of effects, including the intracellular accumulation of cAMP, the mobilization of intracellular Ca2+, and the endocytosis of glucagon and its receptor into intracellular vesicles. In this study, we begin to define the functional role of the COOH-terminal tail of the human glucagon receptor in glucagon-stimulated signal transduction and receptor internalization. We have created and expressed in Chinese hamster ovary (CHO) cells five truncation mutants in which the COOH-terminal 24, 56, 62, 67, and 73 amino acids have been removed. Cells expressing relevant truncated receptors were assayed for cell surface expression by immunofluorescence, for ligand-binding properties, for cAMP and Ca2+-mediated signal transduction properties, and for receptor endocytosis. In addition, a mutant receptor containing seven serine-to-alanine mutations in the COOH-terminal tail was studied. Our results reveal the following: 1) a region of the COOH-terminal tail that is required for proper cell surface expression, 2) the COOH-terminal 62 amino acids, which comprise the majority of the tail, are not required for ligand binding, cAMP accumulation, or Ca2+ mobilization, and 3) phosphorylation of the COOH-terminal tail is crucial for glucagon-stimulated receptor endocytosis.
Buggy
J J
JJ
Bayer Corporation, West Haven, Connecticut 06516, USA.
Heurich
R O
RO
MacDougall
M
M
Kelley
K A
KA
Livingston
J N
JN
Yoo-Warren
H
H
Rossomando
A J
AJ
D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D002118:Calcium / Q000502:physiology; D006224:Cricetinae; D003593:Cytoplasm / Q000502:physiology; D004705:Endocytosis; D005934:Glucagon / Q000502:physiology*; D006801:Humans; D008969:Molecular Sequence Data; D018027:Receptors, Glucagon / Q000502:physiology*; D011994:Recombinant Proteins; D017384:Sequence Deletion; D012694:Serine / Q000737:chemistry; D015398:Signal Transduction; D013329:Structure-Activity Relationship
D018027:Receptors, Glucagon; D011994:Recombinant Proteins; D012694:Serine; D005934:Glucagon; D002118:Calcium