Hans Bräuner-Osborne

Different domains of the glucagon and glucagon-like peptide-1 receptors provide the critical determinants of ligand selectivity.

Glucagon and glucagon‐like peptide‐1 (GLP‐1) are homologous peptide hormones with important functions in glucose metabolism. The receptors for glucagon and GLP‐1 are homologous family B G‐protein coupled receptors. The GLP‐1 receptor amino‐terminal extracellular domain is a major determinant of glucagon/GLP‐1 selectivity of the GLP‐1 receptor. However, the divergent residues in glucagon and GLP‐1 that determine specificity for the GLP‐1 receptor amino‐terminal extracellular domain are not known. Less is known about how the glucagon receptor distinguishes between glucagon and GLP‐1. We analysed chimeric glucagon/GLP‐1 peptides for their ability to bind and activate the glucagon receptor, the GLP‐1 receptor and chimeric glucagon/GLP‐1 receptors. The chimeric peptide GLP‐1(7–20)/glucagon(15–29) was unable to bind and activate the glucagon receptor. Substituting the glucagon receptor core domain with the GLP‐1 receptor core domain (chimera A) completely rescued the affinity and potency of GLP‐1(7–20)/glucagon(15–29) without compromising the affinity and potency of glucagon. Substituting transmembrane segment 1 (TM1), TM6, TM7, the third extracellular loop and the intracellular carboxy‐terminus of chimera A with the corresponding glucagon receptor segments re‐established the ability to distinguish GLP‐1(7–20)/glucagon(15–29) from glucagon. Corroborant results were obtained with the opposite chimeric peptide glucagon(1–14)/GLP‐1(21–37). The results suggest that the glucagon and GLP‐1 receptor amino‐terminal extracellular domains determine specificity for the divergent residues in the glucagon and GLP‐1 carboxy‐terminals respectively. The GLP‐1 receptor core domain is not a critical determinant of glucagon/GLP‐1 selectivity. Conversely, the glucagon receptor core domain contains two or more sub‐segments which strongly determine specificity for divergent residues in the glucagon amino‐terminus.

Structure, pharmacology and therapeutic prospects of family C G-protein coupled receptors.

Family C of G-protein coupled receptors (GPCRs) from humans is constituted by eight metabotropic glutamate (mGlu(1-8)) receptors, two heterodimeric gamma-aminobutyric acid(B) (GABA(B)) receptors, a calcium-sensing receptor (CaR), three taste (T1R) receptors, a promiscuous L-alpha-amino acid receptor (GPRC6A), and five orphan receptors. Aside from the orphan receptors, the family C GPCRs are characterised by a large amino-terminal domain, which bind the endogenous orthosteric agonists. Recently, a number of allosteric modulators binding to the seven transmembrane domains of the receptors have also been reported. Family C GPCRs regulate a number of important physiological functions and are thus intensively pursued as drug targets. So far, two drugs acting at family C receptors (the GABA(B) agonist baclofen and the positive allosteric CaR modulator cinacalcet) have been marketed. Cinacalcet is the first allosteric GPCR modulator to enter the market, which demonstrates that the therapeutic principle of allosteric modulation can also be extended to this important drug target class. In this review we outline the structure and function of family C GPCRs with particular focus on the ligand binding sites, and we present the most important pharmacological agents and the therapeutic prospects of the receptors.

The Agonist-binding Domain of the Calcium-sensing Receptor Is Located at the Amino-terminal Domain

The calcium-sensing receptor (CaR) is a G-protein-coupled receptor that displays 19–25% sequence identity to the γ-aminobutyric acid type B (GABAB) and metabotropic glutamate (mGlu) receptors. All three groups of receptors have a large amino-terminal domain (ATD), which for the mGlu receptors has been shown to bind the endogenous agonist. To investigate whether the agonist-binding domain of the CaR also is located in the ATD, we constructed a chimeric receptor named Ca/1a consisting of the ATD of CaR and the seven transmembrane region and C terminus of mGlu1a. The Ca/1a receptor stimulated inositol phosphate production when exposed to the cationic agonists Ca2+, Mg2+, and Ba2+ in transiently transfected tsA cells (a transformed HEK 293 cell line). The pharmacological profile of Ca/1a (EC50 values of 3.3, 2.6, and 3.9 mm for these cations, respectively) was very similar to that of the wild-type CaR (EC50 values of 3.2, 4.7, and 4.1 mm, respectively). For the mGlu1a receptor, it has been shown that Ser-165 and Thr-188, which are located in the ATD, are involved in the agonist binding. An alignment of CaR with the mGlu receptors showed that these two amino acid residues have been conserved in CaR as Ser-147 and Ser-170, respectively. Each of these residues was mutated to alanines and tested pharmacologically using the endogenous agonist Ca2+. CaR-S147A showed an impaired function as compared with wild-type CaR both with respect to potency of Ca2+(4-fold increase in EC50) and maximal response (79% of wild-type response). CaR-S170A showed no significant response to Ca2+ even at 50 mm concentration. In contrast, each of the two adjacent mutations, S169A and S171A, resulted in pharmacological profiles almost identical to that of the wild-type receptor. These data demonstrate that Ser-170 and to some extent Ser-147 are involved in the Ca2+ activation of the CaR, and taken together, our results reveal a close resemblance of the activation mechanism between the CaR and the mGlu receptors.

Agonists and Inverse Agonists for the Herpesvirus 8-encoded Constitutively Active Seven-transmembrane Oncogene Product, ORF-74

A number of CXC chemokines competed with similar, nanomolar affinity against 125I-interleukin-8 (IL-8) binding to ORF-74, a constitutively active seven-transmembrane receptor encoded by human herpesvirus 8. However, in competition against 125I-labeled growth-related oncogene (GRO)-α, the ORF-74 receptor was highly selective for GRO peptides, with IL-8 being 10,000-fold less potent. The constitutive stimulating activity of ORF-74 on phosphatidylinositol turnover was not influenced by, for example, IL-8 binding. In contrast, GRO peptides acted as potent agonists in stimulating ORF-74 signaling, whereas IP-10 and stromal cell-derived factor-1α surprisingly acted as inverse agonists. These peptides had similar pharmacological properties with regard to enhancing or inhibiting, respectively, the stimulatory effect of ORF-74 on NIH-3T3 cell proliferation. Construction of a high affinity zinc switch through introduction of two His residues at the extracellular end of transmembrane segment V enabled Zn2+ to act as a prototype non-peptide inverse agonist, which eliminated the constitutive signaling. It is concluded that ORF-74, which is believed to be causally involved in the formation of highly vascularized tumors, has been optimized for agonist and inverse agonist modulation by the endogenous angiogenic GRO peptides and angiostatic IP-10 and stromal cell-derived factor-1α, respectively. ORF-74 could serve as a target for the development of non-peptide inverse agonist drugs as demonstrated by the effect of Zn2+ on the metal ion site-engineered receptor.

Extracellular Ca2+ is a danger signal activating the NLRP3 inflammasome through G protein-coupled calcium sensing receptors.

Activation of the NLRP3 inflammasome enables monocytes and macrophages to release high levels of interleukin-1β during inflammatory responses. Concentrations of extracellular calcium can increase at sites of infection, inflammation or cell activation. Here we show that increased extracellular calcium activates the NLRP3 inflammasome via stimulation of G protein-coupled calcium sensing receptors. Activation is mediated by signalling through the calcium-sensing receptor and GPRC6A via the phosphatidyl inositol/Ca2+ pathway. The resulting increase in the intracellular calcium concentration triggers inflammasome assembly and Caspase-1 activation. We identified necrotic cells as one source for excess extracellular calcium triggering this activation. In vivo, increased calcium concentrations can amplify the inflammatory response in the mouse model of carrageenan-induced footpad swelling, and this effect was inhibited in GPRC6A−/− mice. Our results demonstrate that G-protein-coupled receptors can activate the inflammasome, and indicate that increased extracellular calcium has a role as a danger signal and amplifier of inflammation.

Positive allosteric modulation of the human metabotropic glutamate receptor 4 (hmGluR4) by SIB‐1893 and MPEP

We have identified 2‐methyl‐6‐(2‐phenylethenyl)pyridine (SIB‐1893) and 2‐methyl‐6‐phenylethynyl pyridine hydrochloride (MPEP) as positive allosteric modulators for the hmGluR4. SIB‐1893 and MPEP enhanced the potency and efficacy of L‐2‐amino‐4‐phophonobutyrate (L‐AP4) in guanosine 5′‐O‐(3‐[35S]thiotriphosphate ([35S]GTPγS) binding and efficacy in cAMP studies. These effects were fully blocked by the mGluR4 competitive antagonist (RS)‐α‐cyclopropyl‐4‐phosphonophenylglycine (CPPG), indicating a dependency on receptor activation. Although SIB‐1893 and MPEP had no effects alone in GTPγS binding, effects were observed in the cell‐based cAMP assay due to media‐derived activation as indicated by CPPG inhibition. Positive modulation of the mGluR4 was a receptor‐specific effect since SIB‐1893 and MPEP had neither effects on mGluR2‐expressing cells nor on the parent BHK cell line. In [3H]L‐AP4 binding, a two‐fold decrease in KD but not in Bmax was observed with 100 μM SIB‐1893, whereas MPEP affected neither parameter. Finally, SIB‐1893 and MPEP failed to displace [3H]L‐AP4 binding. Taken together, these data identify positive allosteric modulators for the hmGluR4.

Specific γ‐hydroxybutyrate‐binding sites but loss of pharmacological effects of γ‐hydroxybutyrate in GABAB(1)‐deficient mice

γ‐Hydroxybutyrate (GHB), a metabolite of γ‐aminobutyric acid (GABA), is proposed to function as a neurotransmitter or neuromodulator. γ‐Hydroxybutyrate and its prodrug, γ‐butyrolactone (GBL), recently received increased public attention as they emerged as popular drugs of abuse. The actions of GHB/GBL are believed to be mediated by GABAB and/or specific GHB receptors, the latter corresponding to high‐affinity [3H]GHB‐binding sites coupled to G‐proteins. To investigate the contribution of GABAB receptors to GHB actions we studied the effects of GHB in GABAB(1)−/− mice, which lack functional GABAB receptors. Autoradiography reveals a similar spatial distribution of [3H]GHB‐binding sites in brains of GABAB(1)−/− and wild‐type mice. The maximal number of binding sites and the KD values for the putative GHB antagonist [3H]6,7,8,9‐tetrahydro‐5‐hydroxy‐5H‐benzocyclohept‐6‐ylidene acetic acid (NCS‐382) appear unchanged in GABAB(1)−/− compared with wild‐type mice, demonstrating that GHB‐ are distinct from GABAB‐binding sites. In the presence of the GABAB receptor positive modulator 2,6‐di‐tert‐butyl‐4‐(3‐hydroxy‐2,2‐dimethyl‐propyl)‐phenol GHB induced functional GTPγ[35S] responses in brain membrane preparations from wild‐type but not GABAB(1)−/− mice. The GTPγ[35S] responses in wild‐type mice were blocked by the GABAB antagonist [3‐[[1‐(S)‐(3,4dichlorophenyl)ethyl]amino]‐2‐(S)‐hydroxy‐propyl]‐cyclohexylmethyl phosphinic acid hydrochloride (CGP54626) but not by NCS‐382. Altogether, these findings suggest that the GHB‐induced GTPγ[35S] responses are mediated by GABAB receptors. Following GHB or GBL application, GABAB(1)−/− mice showed neither the hypolocomotion, hypothermia, increase in striatal dopamine synthesis nor electroencephalogram delta‐wave induction seen in wild‐type mice. It, therefore, appears that all studied GHB effects are GABAB receptor dependent. The molecular nature and the signalling properties of the specific [3H]GHB‐binding sites remain elusive.

Molecular Pharmacology of Promiscuous Seven Transmembrane Receptors Sensing Organic Nutrients

A number of highly promiscuous seven transmembrane (7TM) receptors have been cloned and characterized within the last few years. It is noteworthy that many of these receptors are activated broadly by amino acids, proteolytic degradation products, carbohydrates, or free fatty acids and are expressed in taste tissue, the gastrointestinal tract, endocrine glands, adipose tissue, and/or kidney. These receptors thus hold the potential to act as sensors of food intake, regulating, for example, release of incretin hormones from the gut, insulin/glucagon from the pancreas, and leptin from adipose tissue. The promiscuous tendency in ligand recognition of these receptors is in contrast to the typical specific interaction with one physiological agonist seen for most receptors, which challenges the classic “lock-and-key” concept. We here review the molecular mechanisms of nutrient sensing of the calcium-sensing receptor, the G protein-coupled receptor family C, group 6, subtype A (GPRC6A), and the taste1 receptor T1R1/T1R3, which are sensing l-α-amino acids, the carbohydrate-sensing T1R2/T1R3 receptor, the proteolytic degradation product sensor GPR93 (also termed GPR92), and the free fatty acid (FFA) sensing receptors FFA1, FFA2, FFA3, GPR84, and GPR120. The involvement of the individual receptors in sensing of food intake has been validated to different degrees because of limited availability of specific pharmacological tools and/or receptor knockout mice. However, as a group, the receptors represent potential drug targets, to treat, for example, type II diabetes by mimicking food intake by potent agonists or positive allosteric modulators. The ligand-receptor interactions of the promiscuous receptors of organic nutrients thus remain an interesting subject of emerging functional importance.

Molecular basis for amino acid sensing by family C G-protein-coupled receptors

Family C of human G‐protein‐coupled receptors (GPCRs) is constituted by eight metabotropic glutamate receptors, two γ‐aminobutyric acid type B (GABAB1–2) subunits forming the heterodimeric GABAB receptor, the calcium‐sensing receptor, three taste1 receptors (T1R1–3), a promiscuous L‐α‐amino acid receptor G‐protein‐coupled receptor family C, group 6, subtype A (GPRC6A) and seven orphan receptors. Aside from the orphan receptors, the family C GPCRs are dimeric receptors characterized by a large extracellular Venus flytrap domain which bind the endogenous agonists. Except from the GABAB1–2 and T1R2–3 receptor, all receptors are either activated or positively modulated by amino acids. In this review, we outline mutational, biophysical and structural studies which have elucidated the interaction of the amino acids with the Venus flytrap domains, molecular mechanisms of receptor selectivity and the initial steps in receptor activation.

α4βδ GABA(A) receptors are high-affinity targets for γ-hydroxybutyric acid (GHB).

γ-Hydroxybutyric acid (GHB) binding to brain-specific high-affinity sites is well-established and proposed to explain both physiological and pharmacological actions. However, the mechanistic links between these lines of data are unknown. To identify molecular targets for specific GHB high-affinity binding, we undertook photolinking studies combined with proteomic analyses and identified several GABAA receptor subunits as possible candidates. A subsequent functional screening of various recombinant GABAA receptors in Xenopus laevis oocytes using the two-electrode voltage clamp technique showed GHB to be a partial agonist at αβδ- but not αβγ-receptors, proving that the δ-subunit is essential for potency and efficacy. GHB showed preference for α4 over α(1,2,6)-subunits and preferably activated α4β1δ (EC50 = 140 nM) over α4β(2/3)δ (EC50 = 8.41/1.03 mM). Introduction of a mutation, α4F71L, in α4β1(δ)-receptors completely abolished GHB but not GABA function, indicating nonidentical binding sites. Radioligand binding studies using the specific GHB radioligand [3H](E,RS)-(6,7,8,9-tetrahydro-5-hydroxy-5H-benzocyclohept-6-ylidene)acetic acid showed a 39% reduction (P = 0.0056) in the number of binding sites in α4 KO brain tissue compared with WT controls, corroborating the direct involvement of the α4-subunit in high-affinity GHB binding. Our data link specific GHB forebrain binding sites with α4-containing GABAA receptors and postulate a role for extrasynaptic α4δ-containing GABAA receptors in GHB pharmacology and physiology. This finding will aid in elucidating the molecular mechanisms behind the proposed function of GHB as a neurotransmitter and its unique therapeutic effects in narcolepsy and alcoholism.