Abstract
The glucagon receptor is a member of a distinct class of G protein-coupled receptors (GPCRs) sharing little amino acid sequence homology with the larger rhodopsin-like GPCR family. To identify the components of the glucagon receptor necessary for G-protein coupling, we replaced sequentially all or part of each intracellular loop (i1, i2, and i3) and the C-terminal tail of the glucagon receptor with the 11 amino acids comprising the first intracellular loop of the D4 dopamine receptor. When expressed in transiently transfected COS-1 cells, the mutant receptors fell into two different groups with respect to hormone-mediated signaling. The first group included the loop i1 mutants, which bound glucagon and signaled normally. The second group comprised the loop i2 and i3 chimeras, which caused no detectable adenylyl cyclase activation in COS-1 cells. However, when expressed in HEK 293T cells, the loop i2 or i3 chimeras caused very small glucagon-mediated increases in cAMP levels and intracellular calcium concentrations, with EC50 values nearly 100-fold higher than those measured for wild-type receptor. Replacement of both loops i2 and i3 simultaneously was required to completely abolish G protein signaling as measured by both cAMP accumulation and calcium flux assays. These results show that the i2 and i3 loops play a role in glucagon receptor signaling, consistent with recent models for the mechanism of activation of G proteins by rhodopsin-like GPCRs.
Highlights
The peptide hormone glucagon plays a pivotal role in the maintenance of metabolic homeostasis
Five mutant receptors were constructed in which the first intracellular loop of the human D4 dopamine receptor was substituted for one or more portions of the cytoplasmic domain of the rat glucagon receptor
To determine the components of the intracellular domain of the glucagon receptor required for proper biosynthesis and signal transduction, we prepared a series of eight glucagon/D4 dopamine receptor chimeras
Summary
Construction of the Rat Glucagon Receptor Mutants—The construction of the synthetic rat glucagon receptor gene (GenBankTM/EMBL Data Bank accession number U14012) was previously reported [11, 17]. The four loop i1 receptor chimeras (G1D1, G1D1n, G1D1i, and G1D1c) and the point mutant H178R, were prepared by replacing a 76-base pair BssHII-BstEII restriction fragment with synthetic duplexes that encoded the desired amino acid residues. An 82-base pair SpeI-NsiI restriction fragment was replaced with a synthetic duplex in which base pairs coding for amino acid residues Ser252-Glu261 of the glucagon receptor were substituted with a sequence coding for the first intracellular loop of the D4 dopamine receptor (TERALQTPTNS). The C terminus chimera, GCD1, was prepared by replacement of a 63-base pair AlwNI-BspEI restriction fragment with a synthetic duplex in which codons for amino acid residues Glu407-Arg415 were substituted with a sequence encoding the first intracellular loop of the D4 dopamine receptor (TERALQTPTNS) followed by two successive stop codons. The IC50 and EC50 values were calculated from the inflection point (c) of the best-fit curve (Sigmaplot, Jandel Scientific Software)
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