Real-time trafficking and signaling of the glucagon-like peptide-1 receptor

The glucagon-like peptide-1 incretin receptor (GLP-1R) of family B G protein-coupled receptors (GPCRs) is a major drug target in type-2-diabetes due to its regulatory effect on post-prandial blood-glucose levels. The mechanism(s) controlling GLP-1R mediated signaling are far from fully understood. A fundamental mechanism controlling the signaling capacity of GPCRs is the post-endocytic trafficking of receptors between recycling and degradative fates. Here, we combined microscopy with novel real-time assays to monitor both receptor trafficking and signaling in living cells. We find that the human GLP-1R internalizes rapidly and with similar kinetics in response to equipotent concentrations of GLP-1 and the stable GLP-1 analogues exendin-4 and liraglutide. Receptor internalization was confirmed in mouse pancreatic islets. GLP-1R is shown to be a recycling receptor with faster recycling rates mediated by GLP-1 as compared to exendin-4 and liraglutide. Furthermore, a prolonged cycling of ligand-activated GLP-1Rs was observed and is suggested to be correlated with a prolonged cAMP signal.     

Beta-Arrestin-1 mediates glucagon-like peptide-1 signaling to insulin secretion in cultured pancreatic beta cells

Glucagon-like peptide-1 (GLP-1) is a polypeptide hormone secreted from enteroendocrine L cells and potentiates glucose-dependent insulin secretion in pancreatic beta cells. Recently the GLP-1 receptor (GLP-1 R) has been a focus for new anti-diabetic therapy with the introduction of GLP-1 analogues and DPP-IV inhibitors, and this has stimulated additional interest in the mechanisms of GLP-1 signaling. Here we identify a mechanism for GLP-1 action, showing that the scaffold protein beta-arrestin-1 mediates the effects of GLP-1 to stimulate cAMP production and insulin secretion in beta cells. Using a coimmunoprecipitation technique, we also found a physical association between the GLP-1 R and beta-arrestin-1 in cultured INS-1 pancreatic beta cells. beta-Arrestin-1 knockdown broadly attenuated GLP-1 signaling, causing decreased ERK and CREB activation and IRS-2 expression as well as reduced cAMP levels and impaired insulin secretion. However, beta-arrestin-1 knockdown did not affect GLP-1 R surface expression and ligand-induced GLP-1 R internalization/desensitization. Taken together, these studies indicate that beta-arrestin-1 plays a role in GLP-1 signaling leading to insulin secretion, defining a previously undescribed mechanism for GLP-1 action.     

Small-molecule agonists for the glucagon-like peptide 1 receptor

The peptide hormone glucagon-like peptide (GLP)-1 has important actions resulting in glucose lowering along with weight loss in patients with type 2 diabetes. As a peptide hormone, GLP-1 has to be administered by injection. Only a few small-molecule agonists to peptide hormone receptors have been described and none in the B family of the G protein coupled receptors to which the GLP-1 receptor belongs. We have discovered a series of small molecules known as ago-allosteric modulators selective for the human GLP-1 receptor. These compounds act as both allosteric activators of the receptor and independent agonists. Potency of GLP-1 was not changed by the allosteric agonists, but affinity of GLP-1 for the receptor was increased. The most potent compound identified stimulates glucose-dependent insulin release from normal mouse islets but, importantly, not from GLP-1 receptor knockout mice. Also, the compound stimulates insulin release from perfused rat pancreas in a manner additive with GLP-1 itself. These compounds may lead to the identification or design of orally active GLP-1 agonists.     

Melanocortin 5 receptor signaling and internalization: Role of MAPK/ERK pathway and β-arrestins 1/2

The Melanocortin 5 receptor (MC5R) is a G-protein coupled receptor (GPCR) that exhibits high affinity for α-MSH. Here we present evidence for MC5R-GFP internalization and subsequent recycling to cell surface, in α-MSH-stimulated HeLa cells. This melanocortin induces a biphasic activation of ERK1/2 with an early peak at 15min, a G(i)-protein driven, β-arrestins 1/2 independent process, and a late sustained activation that is regulated by β-arrestins 1/2. ERK1/2 lead to downstream phosphorylation of 90-kDa ribosomal S6 kinases (p90RSK) and mitogen- and stress-activated protein kinase 1 (MSK1). Only a small fraction (10%) of phosphorylated p90RSK and ERK1/2 translocates to the nucleus inducing c-Fos expression. α-MSH also activates CREB through cAMP/PKA pathway. In 3T3-L1 adipocytes, where MC5R is endogenously expressed, α-MSH also induces phosphorylation and cytosolic retention of the same signaling molecules. These findings provide new evidence on the signaling mechanisms underlying MC5R biological response to α-MSH.     

Absence of the glucagon-like peptide-1 receptor does not affect the metabolic phenotype of mice with liver-specific G(s)α deficiency

The stimulatory G protein α-subunit (G(s)α) couples hormone and other receptors to the generation of intracellular cAMP. We previously showed that mice with liver-specific G(s)α deficiency [liver-specific G(s)α knockout (LGsKO) mice] had reduced adiposity and improved glucose tolerance associated with increased glucose-stimulated insulin secretion, pancreatic islet hyperplasia, and very high serum glucagon and glucagon-like peptide 1 (GLP-1) levels. Because GLP-1 is known to stimulate insulin secretion and to have effects on energy balance, we mated LGsKO mice with germline GLP-1 receptor (GLP-1R) knockout mice (Glp1r(-/-)) and compared LGsKO to double-knockout (LGs/Glp1r(-/-)) mice to determine the contribution of excess GLP-1R signaling to the LGsKO phenotype. Loss of the GLP-1R failed to reverse most of the metabolic features of LGsKO mice, including reduced fat mass, increased glucose tolerance, and second-phase glucose-stimulated insulin secretion, islet cell hyperplasia, and very high glucagon and GLP-1 levels. However, loss of GLP-1R impaired first-phase insulin secretion in mice with or without liver-specific G(s)α deficiency. Thus, excess GLP-1 action (or at least through GLP-1R) does not contribute to the LGsKO metabolic phenotype, and other unknown factors involved in the cross talk between the liver G(s)α/cAMP pathway and pancreatic islet function need to be further elucidated.     

Glucagon-like peptide-1 receptor activation inhibits growth and augments apoptosis in murine CT26 colon cancer cells

Obesity, accompanying or independent of type 2 diabetes mellitus (T2DM), is associated with higher rates of malignancy. Hence, there is considerable interest in understanding whether therapies used to treat obese patients with T2DM impact cancer cell growth. Glucagon-like peptide-1 (GLP-1) is produced in enteroendocrine cells and secreted after meal ingestion. GLP-1 regulates blood glucose through multiple mechanisms, principally inhibition of glucagon and stimulation of insulin secretion. GLP-1 also exerts independent effects promoting cell growth and survival, and sustained activation of GLP-1 receptor (GLP-1R) signaling in rodent thyroid glands leads to C-cell hyperplasia and medullary thyroid cancer. Hence, whether therapies based on GLP-1R activation modify growth or survival of cancer cells is of ongoing interest. We studied the biological actions of GLP-1 in mouse CT26 colon cancer cells that express a functional GLP-1R. The GLP-1R agonist exendin (Ex)-4 (exenatide) increased intracellular cAMP levels and inhibited the activity of signaling kinases glycogen synthase kinase 3 and ERK1/2 in CT26 cells. The Ex-4-induced inactivation of glycogen synthase kinase 3, but not ERK1/2, was dependent on protein kinase A and blocked by the GLP-1R antagonist Ex(9-39). Furthermore, Ex-4 altered cell morphology, induced apoptosis, and inhibited proliferation of CT26 cells in vitro. Moreover Ex-4 decreased CT26 colony formation in soft agar and augmented apoptosis induced by irinotecan. Twice-daily treatment of CT26 tumor-bearing BALB/c mice with Ex-4 for 2 wk increased tumor apoptosis. Hence, GLP-1R activation reduces growth and survival in CT26 colon cancer cells that express the endogenous classical GLP-1R.     

Prototypic G protein-coupled receptor for the intestinotrophic factor glucagon-like peptide 2

Glucagon-like peptide 2 (GLP-2) is a 33-aa proglucagon-derived peptide produced by intestinal enteroendocrine cells. GLP-2 stimulates intestinal growth and up-regulates villus height in the small intestine, concomitant with increased crypt cell proliferation and decreased enterocyte apoptosis. Moreover, GLP-2 prevents intestinal hypoplasia resulting from total parenteral nutrition. However, the mechanism underlying these actions has remained unclear. Here we report the cloning and characterization of cDNAs encoding rat and human GLP-2 receptors (GLP-2R), a G protein-coupled receptor superfamily member expressed in the gut and closely related to the glucagon and GLP-1 receptors. The human GLP-2R gene maps to chromosome 17p13.3. Cells expressing the GLP-2R responded to GLP-2, but not GLP-1 or related peptides, with increased cAMP production (EC₅₀ = 0.58 nM) and displayed saturable high-affinity radioligand binding (K_d = 0.57 nM), which could be displaced by synthetic rat GLP-2 (K_i = 0.06 nM). GLP-2 analogs that activated GLP-2R signal transduction in vitro displayed intestinotrophic activity in vivo. These results strongly suggest that GLP-2, like glucagon and GLP-1, exerts its actions through a distinct and specific novel receptor expressed in its principal target tissue, the gastrointestinal tract.     

Characterization of glucagon-like peptide-1 receptor-binding determinants

Glucagon-like peptide 1 (GLP-1) is a potent insulinotropic hormone currently under study as a therapeutic agent for type 2 diabetes. Since an understanding of the molecular mechanisms leading to high-affinity receptor (R) binding and activation may facilitate the development of more potent GLP-1R agonists, we have localized specific regions of GLP-1R required for binding. The purified N-terminal fragment (hereafter referred to as NT) of the GLP-1R produced in either insect (Sf9) or mammalian (COS-7) cells was shown to bind GLP-1. The physical interaction of NT with GLP-1 was first demonstrated by cross-linking ((125)I-GLP-1/NT complex band at approximately 28 kDa) and secondly by attachment to Ni(2+)-NTA beads. The GLP-1R NT protein attached to beads bound GLP-1, but with lower affinity (inhibitory concentration (IC(50)): 4.5 x 10(-7) M) than wild-type (WT) GLP-1R (IC(50): 5.2 x 10(-9)M). The low affinity of GLP-1R NT suggested that other receptor domains may contribute to GLP-1 binding. This was supported by studies using chimeric glucose-dependent insulinotropic polypeptide (GIP)/GLP-1 receptors. GIP(1-151)/GLP-1R, but not GIP(1-222)/GLP-1R, exhibited specific GLP-1 binding and GLP-1-induced cAMP production, suggesting that the region encompassing transmembrane (TM) domain 1 through to TM3 was required for binding. Since it was hypothesized that certain charged or polar amino acids in this region might be involved in binding, these residues (TM2-TM3) were analyzed by substitution mutagenesis. Five mutants (K197A, D198A, K202A, D215A, R227A) displayed remarkably reduced binding affinity. These studies indicate that the NT domain of the GLP-1R is able to bind GLP-1, but charged residues concentrated at the distal TM2/extracellular loop-1 (EC1) interface (K197, D198, K202) and in EC1 (D215 and R227) probably contribute to the binding determinants of the GLP-1R.     

Glucagon-like peptide-1 enhances glucokinase activity in pancreatic β-cells through the association of Epac2 with Rim2 and Rab3A

Glucokinase (GK), which phosphorylates D-glucose, is a major glucose sensor in β-cells for glucose-stimulated insulin secretion (GSIS) and is a promising new drug target for type 2 diabetes (T2D). In T2D, pancreatic β-cells exhibit defective glucose sensitivity, which leads to impaired GSIS. Although glucagon-like peptide-1-(7-36)-amide (GLP-1) is known to enhance β-cell glucose sensitivity, the effect of GLP-1 on GK activity is still unknown. The present study demonstrated that GLP-1 pretreatment for 30 min significantly enhanced GK activity in a glucose-dependent manner, with a lower Michaelis-Menten constant (K(m)) but unchanged maximal velocity (V(max)). Thus, GLP-1 acutely enhanced cellular glucose uptake, mitochondrial membrane potential, and cellular ATP levels in response to glucose in rat INS-1 and native β-cells. This effect of GLP-1 occurred via its G protein-coupled receptor pathway in a cAMP-dependent but protein kinase A-independent manner with evidence of exchange protein activated by cAMP (Epac) involvement. Silencing Epac2, interacting molecule of the small G protein Rab3 (Rim2), or Ras-associated protein Rab3A (Rab3A) significantly blocked the effect of GLP-1. These results suggested that GLP-1 can further potentiate GSIS by enhancing GK activity through the signaling of Epac2 to Rim2 and Rab3A, which is the similar pathway for GLP-1 to potentiate Ca(2+)-dependent insulin granule exocytosis. The present finding may also be an important mechanism of GLP-1 for recovery of GSIS in T2D.     

Predicted structure of agonist-bound glucagon-like peptide 1 receptor,a class B G protein-coupled receptor

The glucagon-like peptide 1 receptor (GLP1R) is a G protein-coupled receptor (GPCR) involved in insulin synthesis and regulation; therefore, it is an important drug target for treatment of diabetes. However, GLP1R is a member of the class B1 family of GPCRs for which there are no experimental structures. To provide a structural basis for drug design and to probe class B GPCR activation, we predicted the transmembrane (TM) bundle structure of GLP1R bound to the peptide Exendin-4 (Exe4; a GLP1R agonist on the market for treating diabetes) using the MembStruk method for scanning TM bundle conformations. We used protein–protein docking methods to combine the TM bundle with the X-ray crystal structure of the 143-aa N terminus coupled to the Exe4 peptide. This complex was subjected to 28 ns of full-solvent, full-lipid molecular dynamics. We find 14 strong polar interactions of Exe4 with GLP1R, of which 8 interactions are in the TM bundle (2 interactions confirmed by mutation studies) and 6 interactions involve the N terminus (3 interactions found in the crystal structure). We also find 10 important hydrophobic interactions, of which 4 interactions are in the TM bundle (2 interactions confirmed by mutation studies) and 6 interactions are in the N terminus (6 interactions present in the crystal structure). Thus, our predicted structure agrees with available mutagenesis studies. We suggest a number of mutation experiments to further validate our predicted structure. The structure should be useful for guiding drug design and can provide a structural basis for understanding ligand binding and receptor activation of GLP1R and other class B1 GPCRs.     

Structural determinants of dual incretin receptor agonism by tirzepatide

Tirzepatide (LY3298176) is a fatty-acid-modified, dual incretin receptor agonist that exhibits pharmacology similar to native GIP at the glucose-dependent insulinotropic polypeptide receptor (GIPR) but shows bias toward cyclic adenosine monophosphate signaling at the glucagon-like peptide-1 receptor (GLP-1R). In addition to GIPR signaling, the pathway bias at the GLP-1R may contribute to the efficacy of tirzepatide at improving glucose control and body weight regulation in type 2 diabetes mellitus. To investigate the structural basis for the differential signaling of tirzepatide, mechanistic pharmacology studies were allied with cryogenic electron microscopy. Here, we report high-resolution structures of tirzepatide in complex with the GIPR and GLP-1R. Similar to the native ligands, tirzepatide adopts an α-helical conformation with the N terminus reaching deep within the transmembrane core of both receptors. Analyses of the N-terminal tyrosine (Tyr1^Tzp) of tirzepatide revealed a weak interaction with the GLP-1R. Molecular dynamics simulations indicated a greater propensity of intermittent hydrogen bonding between the lipid moiety of tirzepatide and the GIPR versus the GLP-1R, consistent with a more compact tirzepatide–GIPR complex. Informed by these analyses, tirzepatide was deconstructed, revealing a peptide structure–activity relationship that is influenced by acylation-dependent signal transduction. For the GIPR, Tyr1^Tzp and other residues making strong interactions within the receptor core allow tirzepatide to tolerate fatty acid modification, yielding an affinity equaling that of GIP. Conversely, high-affinity binding with the extracellular domain of the GLP-1R, coupled with decreased stability from the Tyr1^Tzp and the lipid moiety, foster biased signaling and reduced receptor desensitization. Together, these studies inform the structural determinants underlying the function of tirzepatide.

Designing therapeutic ligands capable of targeting multiple receptor systems offers opportunities to discover more effective treatments for complex diseases, especially for conditions where intervening at more than one signaling pathway may be beneficial. One such molecule is tirzepatide (LY3298176), a 39-amino acid linear peptide possessing agonist activity at both the glucose-dependent insulinotropic polypeptide receptor (GIPR) and the glucagon-like peptide-1 receptor (GLP-1R) (1, 2). The dual agonist nature of this molecule represents a promising therapeutic modality for the treatment of metabolic disorders, such as type 2 diabetes mellitus (T2DM), obesity (including heart failure patients with preserved ejection fraction), and nonalcoholic steatohepatitis. This therapeutic approach is founded on the established clinical efficacy of selective GLP-1R agonists, with the bifunctional concept being informed by the hypothesis that concerted activation of both receptors improves glucose control, energy balance, and lipid storage (3, 4). In addition to the dual pharmacology, maintaining efficacious concentrations of the drug is important for maximizing the benefit of this type of treatment. Therefore, to sustain the actions of tirzepatide, the peptide is conjugated through a lysine located near the middle of the molecule to a C20 fatty diacid moiety via a hydrophilic linker (2). This fatty acid modification enables reversible, noncovalent binding to human serum albumin and thus contributes to a pharmacokinetic profile that enables once-weekly dosing of the drug (1).To date, the clinical development program for tirzepatide has yielded encouraging results, highlighted by data showing improvements in glycemic control and energy metabolism. The efficacy of tirzepatide to effectively lower glucose and body weight in subjects with T2DM was established in a 26-wk phase 2b trial (5). Moreover, post hoc analyses of this trial reported that treatment with tirzepatide demonstrated favorable effects on markers of insulin sensitivity and pancreatic beta cell function and also reduced atherogenic lipid particles (6, 7). Interestingly, the improvement in insulin sensitivity appeared largely independent of changes in body weight (6). These data supported the initiation of a phase 3 clinical development program for tirzepatide, known as SURPASS (8), and in particular, the results reported from SURPASS-2, a 40-wk pivotal trial in subjects with T2DM, indicate the benefit of dual GIP/GLP-1 pharmacology (9). In this head-to-head study against the highest dose of the GLP-1R monoagonist semaglutide that is currently approved, all three doses of tirzepatide delivered superior glucose and weight reductions. The superior clinical efficacy of tirzepatide points to the advantage of adding GIP pharmacology to GLP-1 therapy.Due to the clinical data, determining the mechanisms responsible for the improvement in metabolic control that occur upon treatment with tirzepatide is an area of active investigation. Ex vivo assays using islets isolated from Gipr or Glp-1r knockout mice and glucose tolerance tests performed in both of these models demonstrate that tirzepatide can enhance insulin secretion and reduce hyperglycemia through either receptor (1). Pharmacologically, receptor-specific cyclic adenosine monophosphate (cAMP) accumulation assays show tirzepatide is an imbalanced agonist favoring GIPR over GLP-1R activity; these results align with binding data indicating the affinity of tirzepatide for the GIPR is equal to that of GIP but approximately fivefold weaker than GLP-1 on the GLP-1R (1, 10). The benefit of strong GIPR activity is supported by findings from studies showing that GIPR agonism improves insulin sensitivity by a mechanism independent of weight loss (11). Furthermore, an intact GIP axis in the brain is necessary to achieve the full anorexigenic effect of dual GIPR and GLP-1R agonist treatment (12). In line with both of these findings, the expression of the GIPR in adipose tissue and certain metabolic control centers in the brain likely contribute to the benefit of the GIP component of tirzepatide (3).At the same time, studies characterizing the pharmacology of tirzepatide at the GLP-1R have garnered attention by showing that it displays pathway bias for cAMP signaling over β-arrestin recruitment (10, 13). The significance of this finding is not fully realized but may be substantive in light of reports showing biased analogs of the GLP-1R agonist exenatide are more efficacious than the nonbiased parent molecule in rodent models (14, 15). Together, the prevailing evidence supports a therapeutic benefit of combining potent GIPR agonism with biased GLP-1R signaling, aligning with the promising outcomes of the clinical studies for tirzepatide. Thus, the experiments performed and presented herein were undertaken to better understand the unique ligand-binding and pharmacological characteristics of tirzepatide on both the GIPR and the GLP-1R at the molecular level.     

Pancreatic glucagon-like peptide-1 receptor couples to multiple G proteins and activates mitogen-activated protein kinase pathways in Chinese hamster ovary cells

Chinese hamster ovary (CHO) cells stably expressing the human insulin receptor and the rat glucagon-like peptide-1 (GLP-1) receptor (CHO/GLPR) were used to study the functional coupling of the GLP-1 receptor with G proteins and to examine the regulation of the mitogen-activated protein (MAP) kinase signaling pathway by GLP-1. We showed that ligand activation of GLP-1 receptor led to increased incorporation of GTP-azidoanilide into Gs alpha, Gq/11 alpha, and Gi1,2 alpha, but not Gi3 alpha. GLP-1 increased p38 MAP kinase activity 2.5- and 2.0-fold over the basal level in both CHO/GLPR cells and rat insulinoma cells (RIN 1046-38), respectively. Moreover, GLP-1 induced phosphorylation of the immediate upstream kinases of p38, MKK3/MKK6, in CHO/GLPR and RIN 1046-38 cells. Ligand-stimulated GLP-1 receptor produced 1.45- and 2.7-fold increases in tyrosine phosphorylation of 42-kDa extracellular signal-regulated kinase (ERK) in CHO/GLPR and RIN 1046-38 cells, respectively. In CHO/GLPR cells, these effects of GLP-1 on the ERK and p38 MAP kinase pathways were inhibited by pretreatment with cholera toxin (CTX), but not with pertussis toxin. The combination of insulin and GLP-1 resulted in an additive response (1.6-fold over insulin alone) that was attenuated by CTX. In contrast, the ability of insulin alone to activate these pathways was insensitive to either toxin. Our study indicates a direct coupling between the GLP-1 receptor and several G proteins, and that CTX-sensitive proteins are required for GLP-1-mediated activation of MAP kinases.     

Novel biological action of the dipeptidylpeptidase-IV inhibitor, sitagliptin, as a glucagon-like peptide-1 secretagogue

Glucagon-like peptide-1 (GLP-1) is an incretin hormone secreted into the circulation by the intestinal L cell. The dipeptidylpeptidase-IV (DPP-IV) inhibitor, sitagliptin, prevents GLP-1 degradation and is used in the clinic to treat patients with type 2 diabetes mellitus, leading to improved glycated hemoglobin levels. When the effect of sitagliptin on GLP-1 levels was examined in neonatal streptozotocin rats, a model of type 2 diabetes mellitus, a 4.9 ± 0.9-fold increase in basal and 3.6 ± 0.4-fold increase in oral glucose-stimulated plasma levels of active GLP-1 was observed (P < 0.001), in association with a 1.5 ± 0.1-fold increase in the total number of intestinal L cells (P < 0.01). The direct effects of sitagliptin on GLP-1 secretion and L cell signaling were therefore examined in murine GLUTag (mGLUTag) and human hNCI-H716 intestinal L cells in vitro. Sitagliptin (0.1-2 μM) increased total GLP-1 secretion by mGLUTag and hNCI-H716 cells (P < 0.01-0.001). However, MK0626 (1-50 μM), a structurally unrelated inhibitor of DPP-IV, did not affect GLP-1 secretion in either model. Treatment of mGLUTag cells with the GLP-1 receptor agonist, exendin-4, did not modulate GLP-1 release, indicating the absence of feedback effects of GLP-1 on the L cell. Sitagliptin increased cAMP levels (P < 0.01) and ERK1/2 phosphorylation (P < 0.05) in both mGLUTag and hNCI-H716 cells but did not alter either intracellular calcium or phospho-Akt levels. Pretreatment of mGLUTag cells with protein kinase A (H89 and protein kinase inhibitor) or MAPK kinase-ERK1/2 (PD98059 and U0126) inhibitors prevented sitagliptin-induced GLP-1 secretion (P < 0.05-0.01). These studies demonstrate, for the first time, that sitagliptin exerts direct, DPP-IV-independent effects on intestinal L cells, activating cAMP and ERK1/2 signaling and stimulating total GLP-1 secretion.     

Positive and negative allosteric modulators promote biased signaling at the calcium-sensing receptor

The calcium-sensing receptor (CaSR) is a G protein-coupled receptor whose function can be allosterically modulated in a positive or negative manner by calcimimetics or calcilytics, respectively. Indeed, the second-generation calcimimetic, cinacalcet, has proven clinically useful in the treatment of chronic kidney disease patients with secondary hyperparathyroidism but is not widely used in earlier stages of renal disease due to the potential to predispose such patients to hypocalcaemia and hyperphosphatemia. The development of a biased CaSR ligand that is more selective for specific signaling pathway(s) leading only to beneficial effects may overcome this limitation. The detection of such stimulus-bias at a G protein-coupled receptor requires investigation across multiple signaling pathways and the development of methods to quantify the effects of allosteric ligands on orthosteric ligand affinity and cooperativity at each pathway. In the current study, we determined the effects of the calcimimetics, NPS-R568 or cinacalcet, and the calcilytic, NPS-2143, on Ca(o)(2+)-mediated intracellular Ca(2+) mobilization, ERK1/2 phosphorylation, and plasma membrane ruffling in a stably transfected human embryonic kidney 293-TREx c-myc-CaSR cell line and applied a novel analytical model to quantify these modulator effects. We present quantitative evidence for the generation of stimulus bias by both positive and negative allosteric modulators of the CaSR, manifested as greater allosteric modulation of intracellular Ca(2+) mobilization relative to ERK1/2 phosphorylation, and a higher affinity of the modulators for the state of the CaSR mediating plasma membrane ruffling relative to the other two pathways. Our findings provide the first evidence that an allosteric modulator used in clinical practice exhibits stimulus bias.     

Functional characterization and structural modeling of obesity associated mutations in the melanocortin 4 receptor

Mutations in the melanocortin 4 receptor (MC4R) gene are the most common known cause of monogenic human obesity. The MC4R gene was sequenced in 2000 subjects with severe early-onset obesity. We detected seven different nonsense and 19 nonsynonymous mutations in a total of 94 probands, some of which have been reported previously by others. We functionally characterized the 11 novel obesity associated missense mutations. Seven of these mutants (L54P, E61K, I69T, S136P, M161T, T162I, and I269N) showed impaired cell surface trafficking, reduced level of maximal binding of the radioligand [125I]NDP-MSH, and reduced ability to generate cAMP in response to ligand. Four mutant MC4Rs (G55V, G55D, S136F, and A303T) displayed cell surface expression and agonist binding similar to the wild-type receptor but showed impaired cAMP production, suggesting that these residues are likely to be critical for conformational rearrangement essential for receptor activation. Homology modeling of these mutants using a model of MC4R based on the crystal structure of the beta2-adrenoreceptor was used to provide insights into the possible structural basis for receptor dysfunction. Transmembrane (TM) domains 1, 3, 6, 7, and peripheral helix 8 appear to participate in the agonist-induced conformational rearrangement necessary for coupling of ligand binding to signaling. We conclude that G55V, G55D, S136F, and A303T mutations are likely to strengthen helix-helix interactions between TM1 and TM2, TM3 and TM6, and TM7 and helix 8, respectively, preventing relative movement of these helices during receptor activation. The combination of functional studies and structural modeling of naturally occurring pathogenic mutations in MC4R can provide valuable information regarding the molecular mechanism of MC4R activation and its dysfunction in human disease.     

MC1R and cAMP signaling inhibit cdc25B activity and delay cell cycle progression in melanoma cells

The melanocortin 1 receptor (MC1R) mediates the tanning response through induction of cAMP and downstream pigmentary enzymes. Diminished function alleles of MC1R are associated with decreased tanning and increased melanoma risk, which has been attributed to increased rates of mutation. We have found that MC1R or cAMP signaling also directly decreases proliferation in melanoma cell lines. MC1R overexpression, treatment with the MC1R ligand, or treatment with small-molecule activators of cAMP signaling causes delayed progression from G2 into mitosis. This delay is caused by phosphorylation and inhibition of cdc25B, a cyclin dependent kinase 1-activating phosphatase, and is rescued by expression of a cdc25B mutant that cannot be phosphorylated at the serine 323 residue. These results show that MC1R and cAMP signaling can directly inhibit melanoma growth through regulation of the G2/M checkpoint.     

Isolation and structure-function studies of a glucagon-like peptide 1 receptor from goldfish Carassius auratus: identification of three charged residues in extracellular domains critical for receptor function

A better understanding of the molecular mechanism of ligand-receptor interaction of glucagon-like peptide 1 (GLP-1) receptors (GLP-1Rs) is useful for the design of potent GLP-1 analogs that could potentially be used as a treatment for diabetic patients. Changes in the ligand and receptor sequences during evolution provide invaluable clues to evaluate the functional motifs of the receptor that are responsible for ligand interaction. For these reasons, in the present study, we have isolated and functionally characterized a GLP-1R from goldfish. Its amino acid sequence shows 50.8% and 52.3% identity with the human glucagon (hGLU) and GLP-1Rs, respectively, and 84.1% with the zebrafish GLP-1R (the only other GLP-1R isolated from teleost fish). Peptides that are structurally different from goldfish (gf)GLP-1, such as gfGLU and hGLU and human GLP-1 (7-36)amide, are also capable of stimulating this receptor, albeit with lower potencies than gfGLP-1. gfGLP-1 stimulates the formation of cAMP through the recombinant gfGLP-1R with EC(50) = 0.18 nM, whereas EC(50) values for gfGLU, human GLP-1 (7-36)amide, and hGLU are 0.53 nM, 0.9 nM, and 1.2 nM, respectively. These results indicate that the gfGLP-1R is structurally more flexible than its mammalian counterpart and that its binding pocket can accommodate a wider spectrum of peptide ligands. Previous studies demonstrated that the charged residues in the extracellular domains of mammalian GLP-1R, particularly those found in the N-terminal domain and the first exoloop, are important for ligand binding. We investigated the roles of the conserved charged residues in the function of the gfGLP-1R. Eleven mutant receptors were constructed, and the effects of mutations were determined by functional assays. Our results demonstrated that three charged residues (D(113), R(197), and D(205)) present in the extracellular domains are critical for receptor function.     

Discovery of β-arrestin-biased dopamine D2 ligands for probing signal transduction pathways essential for antipsychotic efficacy

Elucidating the key signal transduction pathways essential for both antipsychotic efficacy and side-effect profiles is essential for developing safer and more effective therapies. Recent work has highlighted noncanonical modes of dopamine D(2) receptor (D(2)R) signaling via β-arrestins as being important for the therapeutic actions of both antipsychotic and antimanic agents. We thus sought to create unique D(2)R agonists that display signaling bias via β-arrestin-ergic signaling. Through a robust diversity-oriented modification of the scaffold represented by aripiprazole (1), we discovered UNC9975 (2), UNC0006 (3), and UNC9994 (4) as unprecedented β-arrestin-biased D(2)R ligands. These compounds also represent unprecedented β-arrestin-biased ligands for a G(i)-coupled G protein-coupled receptor (GPCR). Significantly, UNC9975, UNC0006, and UNC9994 are simultaneously antagonists of G(i)-regulated cAMP production and partial agonists for D(2)R/β-arrestin-2 interactions. Importantly, UNC9975 displayed potent antipsychotic-like activity without inducing motoric side effects in inbred C57BL/6 mice in vivo. Genetic deletion of β-arrestin-2 simultaneously attenuated the antipsychotic actions of UNC9975 and transformed it into a typical antipsychotic drug with a high propensity to induce catalepsy. Similarly, the antipsychotic-like activity displayed by UNC9994, an extremely β-arrestin-biased D(2)R agonist, in wild-type mice was completely abolished in β-arrestin-2 knockout mice. Taken together, our results suggest that β-arrestin signaling and recruitment can be simultaneously a significant contributor to antipsychotic efficacy and protective against motoric side effects. These functionally selective, β-arrestin-biased D(2)R ligands represent valuable chemical probes for further investigations of D(2)R signaling in health and disease.     

Beta-blockers alprenolol and carvedilol stimulate beta-arrestin-mediated EGFR transactivation

Recent evidence suggests that binding of agonist to its cognate receptor initiates not only classical G protein-mediated signaling, but also beta-arrestin-dependent signaling. One such beta-arrestin-mediated pathway uses the beta(1)-adrenergic receptor (beta(1)AR) to transactivate the EGFR. To determine whether beta-adrenergic ligands that do not activate G protein signaling (i.e., beta-blockers) can stabilize the beta(1)AR in a signaling conformation, we screened 20 beta-blockers for their ability to stimulate beta-arrestin-mediated EGFR transactivation. Here we show that only alprenolol (Alp) and carvedilol (Car) induce beta(1)AR-mediated transactivation of the EGFR and downstream ERK activation. By using mutants of the beta(1)AR lacking G protein-coupled receptor kinase phosphorylation sites and siRNA directed against beta-arrestin, we show that Alp- and Car-stimulated EGFR transactivation requires beta(1)AR phosphorylation at consensus G protein-coupled receptor kinase sites and beta-arrestin recruitment to the ligand-occupied receptor. Moreover, pharmacological inhibition of Src and EGFR blocked Alp- and Car-stimulated EGFR transactivation. Our findings demonstrate that Alp and Car are ligands that not only act as classical receptor antagonists, but can also stimulate signaling pathways in a G protein-independent, beta-arrestin-dependent fashion.