Characterization of Deletion and Truncation Mutants of the Rat Glucagon Receptor

Unson, Cecilia G.; Cypess, Aaron M.; Kim, Hyang Nina; Goldsmith, Paul K.; Carruthers, Christopher; Merrifield, R. B.; Sakmar, Thomas P.

doi:10.1074/jbc.270.46.27720

Cited by 87 publications

(75 citation statements)

References 51 publications

(45 reference statements)

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…H22 cell membranes were incubated with radiolabeled glucagon and increasing concentrations of unlabeled glucagon. The competition binding curves were well described by a logistic function characteristic of ligand-receptor equilibrium binding (data not shown) (7). The concentration of unlabeled glucagon required to displace 50% of receptor-bound 125 I-glucagon (IC 50 value) was 13 nM, which is consistent with previous reports for the GR in both tissue samples and heterologous expression systems (1,6,7).…”

Section: Resultssupporting

confidence: 78%

“…Depending on cell type, cAMP can activate PKA to exert either stimulatory or inhibitory effects on MAP kinase (13)(14)(15)(16)(17). We previously prepared a series of GR mutants that have essentially normal affinity for glucagon but different abilities to produce cAMP (7,9). These mutants were expressed in HEK 293 cells to assess the regulatory effect of cAMP on ERK1͞2 activity.…”

Section: Resultsmentioning

confidence: 99%

“…Clones were selected in the presence of 200 g͞ml G418 (GIBCO͞BRL) and expanded. Clone H22 was selected for stable expression of GR based on specific glucagon binding and glucagon-induced signaling assays (1,6,7). Transient transfection of HEK 293 cells was performed on 80% confluent monolayers in 60-mm plates by using Lipofectamine.…”

Section: Methodsmentioning

confidence: 99%

See 2 more Smart Citations

Glucagon receptor activates extracellular signal-regulated protein kinase 1/2 via cAMP-dependent protein kinase

Jiang

Cypess

Muse

et al. 2001

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

View full text Add to dashboard Cite

We prepared a stable cell line expressing the glucagon receptor to characterize the effect of Gs-coupled receptor stimulation on extracellular signal-regulated protein kinase 1͞2 (ERK1͞2) activity. Glucagon treatment of the cell line caused a dose-dependent increase in cAMP concentration, activation of cAMP-dependent protein kinase (PKA), and transient release of intracellular calcium. Glucagon treatment also caused rapid dose-dependent phosphorylation and activation of mitogen-activated protein kinase kinase͞ ERK kinase (MEK1͞2) and ERK1͞2. Inhibition of either PKA or MEK1͞2 blocked ERK1͞2 activation by glucagon. However, no significant activation of several upstream activators of MEK, including Ras, Rap1, and Raf, was observed in response to glucagon treatment. In addition, chelation of intracellular calcium reduced glucagon-mediated ERK1͞2 activation. In transient transfection experiments, glucagon receptor mutants that bound glucagon but failed to increase intracellular cAMP and calcium concentrations showed no glucagon-stimulated ERK1͞2 phosphorylation. We conclude that glucagon-induced MEK1͞2 and ERK1͞2 activation is mediated by PKA and that an increase in intracellular calcium concentration is required for maximal ERK activation.G lucagon exerts its regulatory effects on hepatic glucose production by binding to the glucagon receptor (GR) (1). GR belongs to the superfamily of heptahelical transmembrane G protein-coupled receptors (GPCRs), which is divided into subfamilies based on amino acid sequence comparison. Most GPCRs fall into family A, the opsin͞adrenergic receptor subfamily. GR is in family B, which shares few amino acid sequence similarities with the other GPCRs. Nevertheless, family B receptors generally couple to the same heterotrimeric G protein classes and activate the same sets of downstream effectors and signaling networks as the family A receptors.One set of downstream effectors is the mitogen-activated protein (MAP) kinases, which include extracellular signalregulated protein kinase 1͞2 (ERK1͞2), p38, and c-Jun Nterminal kinase͞stress-activated protein kinase. These cytoplasmic serine͞threonine protein kinases are critical points of convergence for cellular signal transduction pathways leading to cellular differentiation, proliferation, and transformation (2). ERK1͞2 MAP kinases originally were shown to be activated by receptor tyrosine kinases that relied on a cascade of proteinprotein interactions and phosphorylations proceeding through Ras, Raf, and MAP kinase kinase͞ERK kinase (MEK1͞2) (3). It has since been shown that each of the four families of heterotrimeric G proteins (G s , G i , G q , and G 12 ) activates, or sometimes inhibits, MAP kinase activity (4, 5). Although much attention has been focused on regulation of MAP kinases by family A GPCRs, much less is known about how family B receptors regulate MAP kinase activity.We prepared a clonal cell line from human embryonic kidney (HEK) 293 cells that stably expressed a synthetic gene for the rat GR. Glucagon treatment of the cells ...

show abstract

Section: Resultssupporting

confidence: 78%

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

Glucagon receptor activates extracellular signal-regulated protein kinase 1/2 via cAMP-dependent protein kinase

Jiang

Cypess

Muse

et al. 2001

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

View full text Add to dashboard Cite

show abstract

“…Reports on similar rNT-TM1 constructs showing membrane presence but no detectable binding to their cognate hormones also exist (34,35).…”

Section: Resultsmentioning

confidence: 99%

The Isolated N-terminal Domain of the Glucagon-like Peptide-1 (GLP-1) Receptor Binds Exendin Peptides with Much Higher Affinity than GLP-1

Maturana

Willshaw

Kuntzsch

et al. 2003

Journal of Biological Chemistry

View full text Add to dashboard Cite

It is well known that the action of glucose on pancreatic islets results in increased plasma insulin levels. Nevertheless, high blood glucose levels are not solely responsible for increased insulin secretion (for review, see Ref. 1). For example, in 1964 McIntyre et al. (2) demonstrated that intravenous injection of glucose resulted in a smaller insulin release than that resulting from intrajejunal glucose injection, even though the latter produced lower blood glucose levels compared with the former. Hence, glucose-dependent insulin secretion requires a nutrientdependent component, which was believed to be an endocrine transmitter termed an "incretin" (3). It has since been demonstrated that two hormones, glucagon-like peptide-1 and glucosedependent insulinotropic polypeptide, are responsible for the incretin effect (1).The predominant active form of GLP-1 is actually glucagonlike peptide-1(7-36)amide (termed GLP-1 1 throughout this paper), a 30-residue peptide hormone derived from the post-translational modification of proglucagon in intestinal L cells (1). GLP-1 not only increases glucose-dependent insulin secretion (4 -6), but it also decreases glucose-dependent glucagon secretion (7, 8) and decelerates gastric emptying (9). In addition, GLP-1 has been shown to reduce appetite in rats (10) and to stimulate proinsulin gene transcription and biosynthesis in pancreatic ␤-cells (11, 12). The physiological roles of GLP-1 in maintaining blood sugar levels, via a glucose-dependent mechanism, have heightened interest in the GLP-1 receptor (GLP-1R) as a target for glucose-dependent therapeutic agents designed to treat hyperglycemia resulting from diabetes (13,14). Unfortunately, the half-life of GLP-1 itself after subcutaneous injection is very short because of dipeptidyl peptidase IV cleavage of the first 2 N-terminal residues (15), and so future research requires the design of physiologically stable GLP-1R agonists.The venom of the Gila monster Heloderma suspectum contains a mixture of compounds that includes several peptides related in sequence to GLP-1. Two of these, exendin-3 and exendin-4, are 39-amino acid peptides that share ϳ50% sequence identity to GLP-1 itself and are indeed potent GLP-1R agonists (Fig. 1) (16, 17). Interestingly, although GLP-1 affinity is highly sensitive to N-terminal cleavage, exendin-4 can be truncated by up to 8 residues at its N terminus without significant loss of affinity, suggesting that relative to GLP-1, the central and/or C-terminal residues form additional stabilizing contacts with the receptor (15, 18). Nevertheless, the first two amino acids are also essential for the efficacy of exendin peptides because, once removed, the truncated exendin peptides function as antagonists or inverse agonists (16 -19).

show abstract

“…Membranes from these cells were prepared as previously described. 10,11 Total protein membrane concentration was determined by a modified Lowry method using the Bio-Rad DC protein assay kit (Bio-Rad, Hercules, CA). 20 Deglycosylation of the receptor protein was done by treating an aliquot of membranes with peptide N-glycosidase F (PNGase F) (New England Biolabs, Beverly, MA) in the buffer provided at 378C for 2 h. PNGase F is an amidase that cleaves asparagine-or N-linked oligosaccharides from glycoproteins.…”

Section: Tetr-hgr Cell Lines At Least 25 Colonies Of Hek293s(gn-t1)mentioning

confidence: 99%

Expression of glucagon receptors in tetracycline‐inducible HEK293S GnT1⁻ stable cell lines: An approach toward purification of receptor protein for structural studies

Unson

2008

Biopolymers

Self Cite

View full text Add to dashboard Cite

The nested one‐male units (OMUs) of the hamadryas baboon are part of a complex social system in which “leader” males achieve near exclusive mating access by forcibly herding females into permanent consortships. Within this multi‐level social system (troops, bands, clans and OMUs) are two types of prereproductive males—the follower and solitary male—whose different trajectories converge on the leader role. Here we compare OMU formation strategies of followers, who associate with a particular OMU and may have social access to females, with those of solitary males, who move freely within the band and do not associate regularly with OMUs. Data were derived from 42 OMU formations (16 by followers and 26 by solitary males) occurring over 8 years in a hamadryas baboon band at the Filoha site in Ethiopia. “Initial units” (IUs) with sexually immature females (IU strategy) were formed by 44% of followers and 46% of solitary males. The remaining followers took over mature females when their leader was deposed (challenge strategy) or disappeared (opportunistic strategy), or via a seemingly peaceful transfer (inheritance strategy). Solitary males took over mature females from other clans and bands, but mainly from old, injured or vanished leaders within their clan (via both the challenge and opportunistic strategies). Former followers of an OMU were more successful at taking over females from those OMUs than any other category of male. Despite this advantage enjoyed by ex‐follower leaders, ex‐solitary leaders were equally capable of increasing their OMU size at a comparable rate in their first 2 years as a leader. These results demonstrate the potential for males to employ both multiple roles (follower vs. solitary male) and multiple routes (IU, inheritance, challenge, opportunistic) to acquire females and become a leader male in a mating system characterized by female defense polygyny in a competitive arena. Am. J. Primatol. 73:679–691, 2011. © 2011 Wiley‐Liss, Inc.

show abstract

Characterization of Deletion and Truncation Mutants of the Rat Glucagon Receptor

Cited by 87 publications

References 51 publications

Glucagon receptor activates extracellular signal-regulated protein kinase 1/2 via cAMP-dependent protein kinase

Glucagon receptor activates extracellular signal-regulated protein kinase 1/2 via cAMP-dependent protein kinase

The Isolated N-terminal Domain of the Glucagon-like Peptide-1 (GLP-1) Receptor Binds Exendin Peptides with Much Higher Affinity than GLP-1

Expression of glucagon receptors in tetracycline‐inducible HEK293S GnT1⁻ stable cell lines: An approach toward purification of receptor protein for structural studies

Contact Info

Product

Resources

About

Characterization of Deletion and Truncation Mutants of the Rat Glucagon Receptor

Cited by 87 publications

References 51 publications

Glucagon receptor activates extracellular signal-regulated protein kinase 1/2 via cAMP-dependent protein kinase

Glucagon receptor activates extracellular signal-regulated protein kinase 1/2 via cAMP-dependent protein kinase

The Isolated N-terminal Domain of the Glucagon-like Peptide-1 (GLP-1) Receptor Binds Exendin Peptides with Much Higher Affinity than GLP-1

Expression of glucagon receptors in tetracycline‐inducible HEK293S GnT1− stable cell lines: An approach toward purification of receptor protein for structural studies

Contact Info

Product

Resources

About

Expression of glucagon receptors in tetracycline‐inducible HEK293S GnT1⁻ stable cell lines: An approach toward purification of receptor protein for structural studies