Differential engagement of polar networks in the glucagon-like peptide 1 receptor by endogenous variants of the glucagon-like peptide 1.

The glucagon-like peptide 1 receptor (GLP-1R) can be activated by a number of endogenous peptide hormones, including extended, processed, glycine extended and carboxy-terminally amidated versions of glucagon-like peptide 1 (GLP-1). While the main focus of the literature has been on the processed, amidated form, GLP-1(7-36)NH2, the other forms of this peptide are likely to be secreted in physiologically relevant amounts under certain circumstances. This study builds on our existing work examining the effect of mutation of conserved transmembrane polar residues within the receptor to understand the nature of binding and pleiotropic signaling in response to these alternatively processed versions of this important incretin hormone. We show that extended and processed peptides differ not only in their binding to the receptor but also in the way the receptor is engaged for activation that leads to differential signaling bias exhibited by these peptides.

Structural and functional insights into the juxtamembranous amino-terminal tail and extracellular loop regions of class B GPCRs.

Class B guanine nucleotide-binding protein GPCRs share heptahelical topology and signalling via coupling with heterotrimeric G proteins typical of the entire superfamily of GPCRs. However, they also exhibit substantial structural differences from the more extensively studied class A GPCRs. Even their helical bundle region, most conserved across the superfamily, is predicted to differ from that of class A GPCRs. Much is now known about the conserved structure of the amino-terminal domain of class B GPCRs, coming from isolated NMR and crystal structures, but the orientation of that domain relative to the helical bundle is unknown, and even less is understood about the conformations of the juxtamembranous amino-terminal tail or of the extracellular loops linking the transmembrane segments. We now review what is known about the structure and function of these regions of class B GPCRs. This comes from indirect analysis of structure-function relationships elucidated by mutagenesis and/or ligand modification and from the more direct analysis of spatial approximation coming from photoaffinity labelling and cysteine trapping studies. Also reviewed are the limited studies of structure of some of these regions. No dominant theme was recognized for the structures or functional roles of distinct regions of these juxtamembranous portions of the class B GPCRs. Therefore, it is likely that a variety of molecular strategies can be engaged for docking of agonist ligands and for initiation of conformational changes in these receptors that would be expected to converge to a common molecular mechanism for activation of intracellular signalling cascades.

Molecular mechanisms underlying physiological and receptor pleiotropic effects mediated by GLP-1R activation.

The incidence of type 2 diabetes in developed countries is increasing yearly with a significant negative impact on patient quality of life and an enormous burden on the healthcare system. Current biguanide and thiazolidinedione treatments for type 2 diabetes have a number of clinical limitations, the most serious long-term limitation being the eventual need for insulin replacement therapy (Table 1). Since 2007, drugs targeting the glucagon-like peptide-1 (GLP-1) receptor have been marketed for the treatment of type 2 diabetes. These drugs have enjoyed a great deal of success even though our underlying understanding of the mechanisms for their pleiotropic effects remain poorly characterized even while major pharmaceutical companies actively pursue small molecule alternatives. Coupling of the GLP-1 receptor to more than one signalling pathway (pleiotropic signalling) can result in ligand-dependent signalling bias and for a peptide receptor such as the GLP-1 receptor this can be exaggerated with the use of small molecule agonists. Better consideration of receptor signalling pleiotropy will be necessary for future drug development. This is particularly important given the recent failure of taspoglutide, the report of increased risk of pancreatitis associated with GLP-1 mimetics and the observed clinical differences between liraglutide, exenatide and the newly developed long-acting exenatide long acting release, albiglutide and dulaglutide.

Receptor activity-modifying protein-dependent impairment of calcitonin receptor splice variant Δ(1-47)hCT((a)) function.

BACKGROUND AND PURPOSE: Alternative splicing expands proteome diversity to GPCRs. Distinct receptor variants have been identified for a secretin family GPCR, the calcitonin receptor (CTR). The possible functional contributions of these receptor variants are further altered by their potential interactions with receptor activity-modifying proteins (RAMPs). One variant of the human CTR lacks the first 47 residues at its N terminus [Δ(1-47)hCT((a)) ]. However, very little is known about the pharmacology of this variant or its ability to interact with RAMPs to form amylin receptors. EXPERIMENTAL APPROACH: Δ(1-47)hCT((a)) was characterized both with and without RAMPs in Cos7 and/or HEK293S cells. The receptor expression (ELISA assays) and function (cAMP and pERK1/2 assays) for up to six agonists and two antagonists were determined. KEY RESULTS: Despite lacking 47 residues at the N terminus, Δ(1-47)hCT((a)) was still able to express at the cell surface, but displayed a generalized reduction in peptide potency. Δ(1-47)hCT((a)) retained its ability to interact with RAMP1 and formed a functional amylin receptor; this also appeared to be the case with RAMP3. On the other hand, its interaction with RAMP2 and resultant amylin receptor was reduced to a greater extent. CONCLUSIONS AND IMPLICATIONS: Δ(1-47)hCT((a)) acts as a functional receptor at the cell surface. It exhibits altered receptor function, depending on whether it associates with a RAMP and which RAMP it interacts with. Therefore, the presence of this variant in tissues will potentially contribute to altered peptide binding and signalling, depending on the RAMP distribution in tissues.

Receptor activity modifying proteins (RAMPs) interact with the VPAC2 receptor and CRF1 receptors and modulate their function.

BACKGROUND AND PURPOSE: Although it is established that the receptor activity modifying proteins (RAMPs) can interact with a number of GPCRs, little is known about the consequences of these interactions. Here the interaction of RAMPs with the glucagon-like peptide 1 receptor (GLP-1 receptor), the human vasoactive intestinal polypeptide/pituitary AC-activating peptide 2 receptor (VPAC(2)) and the type 1 corticotrophin releasing factor receptor (CRF(1)) has been examined. EXPERIMENTAL APPROACH: GPCRs were co-transfected with RAMPs in HEK 293S and CHO-K1 cells. Cell surface expression of RAMPs and GPCRs was examined by ELISA. Where there was evidence for interactions, agonist-stimulated cAMP production, Ca(2+) mobilization and GTPγS binding to G(s), G(i), G(12) and G(q) were examined. The ability of CRF to stimulate adrenal corticotrophic hormone release in Ramp2(+/-) mice was assessed. KEY RESULTS: The GLP-1 receptor failed to enhance the cell surface expression of any RAMP. VPAC(2) enhanced the cell surface expression of all three RAMPs. CRF(1) enhanced the cell surface expression of RAMP2; the cell surface expression of CRF(1) was also increased. There was no effect on agonist-stimulated cAMP production. However, there was enhanced G-protein coupling in a receptor and agonist-dependent manner. The CRF(1) : RAMP2 complex resulted in enhanced elevation of intracellular calcium to CRF and urocortin 1 but not sauvagine. In Ramp2(+/-) mice, there was a loss of responsiveness to CRF. CONCLUSIONS AND IMPLICATIONS: The VPAC(2) and CRF(1) receptors interact with RAMPs. This modulates G-protein coupling in an agonist-specific manner. For CRF(1), coupling to RAMP2 may be of physiological significance.

Lifting the lid on GPCRs: the role of extracellular loops.

GPCRs exhibit a common architecture of seven transmembrane helices (TMs) linked by intracellular loops and extracellular loops (ECLs). Given their peripheral location to the site of G-protein interaction, it might be assumed that ECL segments merely link the important TMs within the helical bundle of the receptor. However, compelling evidence has emerged in recent years revealing a critical role for ECLs in many fundamental aspects of GPCR function, which supported by recent GPCR crystal structures has provided mechanistic insights. This review will present current understanding of the key roles of ECLs in ligand binding, activation and regulation of both family A and family B GPCRs.

Extracellular loops and ligand binding to a subfamily of Family A G-protein-coupled receptors.

GPCRs (G-protein-coupled receptors) are a large family of structurally related proteins which mediate their effects by coupling to G-proteins. The V(1a)R (V(1a) vasopressin receptor) is a member of a family of related GPCRs that are activated by vasopressin {AVP ([Arg(8)]vasopressin)}, OT (oxytocin) and related peptides. These receptors are members of a subfamily of Family A GPCRs called the neurohypophysial peptide hormone receptor family. GPCRs exhibit a conserved tertiary structure comprising a bundle of seven TM (transmembrane) helices linked by alternating ECLs (extracellular loops) and ICLs (intracellular loops). The cluster of TM helices is functionally important for ligand binding, and, furthermore, activation of GPCRs involves movement of these TM helices. Consequently, it might be assumed that the extracellular face of GPCRs is composed of peptide linkers that merely connect important TM helices. However, using a systematic mutagenesis approach and focusing on the N-terminus and the second ECL of the V(1a)R, we have established that these extracellular domains fulfil a range of important roles with respect to GPCR signalling, including agonist binding, ligand selectivity and receptor activation.

Concentric and eccentric isokinetic resistance training similarly increases muscular strength, fat-free soft tissue mass, and specific bone mineral measurements in young women.

UNLABELLED: Women participated in 5 months of unilateral concentric (n = 37) or eccentric (n = 33) isokinetic resistance training of the legs and arms. Limb muscular strength increased as did total body, leg, and arm fat-free soft tissue mass, total body BMC, hip BMD, and forearm BMC and BMD. Isokinetic training benefits bone mineral acquisition. INTRODUCTION AND HYPOTHESIS: Isokinetic resistance training (IRT) is osteogenic; however, it is not known if concentric or eccentric modalities of IRT produce differential effects on bone. We tested our hypothesis that high-load eccentric versus concentric mode of IRT would produce greater increases in muscular strength, fat-free soft tissue mass (FFSTM), bone mineral density (BMD) and content (BMC) in trained legs and arms. METHODS: Participants were randomized to 5 months of concentric (n = 37) or eccentric (n = 33) training. The non-dominant leg and arm were used during training; dominant limbs served as controls. Muscular strength was measured with an isokinetic dynamometer; body composition was measured by dual-energy X-ray absorptiometry. RESULTS: Muscular strength of the concentrically and eccentrically trained leg (18.6%; 28.9%) and arm (12.5%; 24.6%) significantly increased with training. Gains in total body (TB) BMC (p < 0.05) and, in the trained limbs, total proximal femur BMD (p < 0.05) and total forearm BMD (p < 0.05) and BMC (p < 0.05) occurred in both groups. FFSTM increased for the TB and trained leg and arm (all p < 0.001) in both modes. CONCLUSION: Regardless of the mode, high-intensity, slow-velocity IRT increases muscular strength and FFSTM of trained limbs and imparts benefits to TB BMC and site-specific BMD and BMC in young women.

Relationships among bone mineral density, body composition, and isokinetic strength in young women.

The purpose of this study was to examine the relationships among bone mineral density (BMD), body composition, and isokinetic strength in young women. Subjects were 76 women (age: 20 +/- 2 yr, height: 164 +/- 6 cm, weight: 57 +/- 6 kg, body fat: 27 +/- 4%) with a normal body mass index (18-25 kg/m(2)). Total body, nondominant proximal femur, and nondominant distal forearm BMD were measured with dual-energy x-ray absorptiometry. Isokinetic concentric (CON) and eccentric (ECC) strength of the nondominant thigh and upper arm were measured at 60 deg/sec. Fat-free mass (FFM) correlated (P < 0.001) with BMD of the total body (r = 0.56) and femoral neck (r = 0.52), whereas fat mass (FM) did not relate to BMD at any site. Leg FFM, but not FM, correlated with BMD in all regions of interest at the proximal femur. Weak associations were observed between arm FFM and forearm BMD. Isokinetic strength did not relate to BMD at any site after correcting for regional FFM. In conclusion, strong, independent associations exist between BMD and FFM, but not FM or isokinetic strength, in young women.

An improved accuracy six-load component pedal dynamometer for cycling.

This paper describes a new six-load component pedal dynamometer designed for study of knee overuse injury in cycling. A unique capability of the dynamometer is the ability to interface with multiple pedal platforms of varying height while maintaining a desired elevation of the foot above the pedal spindle axis. The dynamometer was designed using a concept described in an earlier article by Quinn and Mote (1991, Exp. Mech. 30, 40-48) which measures shear strain across multiple, thin cross-sections. An optimal design technique was used for choosing dimensions of the load measuring cross-sections. A dynamometer was designed and built using the optimal results. Calibration, accuracy results, and sample data are presented. A comparison of accuracy reveals that the new dynamometer is more accurate than previously reported instruments.