Robert G. MacKenzie

Functional Characterization of Mutations in Melanocortin-4 Receptor Associated with Human Obesity

Melanocortin-4 receptor (MC4R) is a G protein-coupled receptor implicated in the regulation of body weight. Genetic studies in humans have identified two frameshift mutations of MC4R associated with a dominantly inherited form of obesity. We have generated and expressed the corresponding MC4R mutants in 293T cells and found that cells transfected with the truncation mutants failed to exhibit agonist binding or responsiveness despite retention of structural motifs potentially sufficient for binding and signaling. Immunofluorescence studies showed that the mutant proteins were expressed and localized in the intracellular compartment but absent from the plasma membrane, suggesting that these mutations disrupted the proper cellular transport of MC4R. Further studies identified a sequence in the cytoplasmic tail of MC4R necessary for the cell surface targeting. We further investigated a possible dominant-negative activity of the mutants on wild-type receptor function. Co-transfection studies showed that the mutants affected neither signaling nor cell surface expression of wild-type MC4R. We also characterized three human sequence variants of MC4R, but these exhibited identical affinities for peptide ligands and identical agonist responsiveness. Thus, unlike the obesity-associated MC4R truncation mutants, the polymorphisms of MC4R are unlikely to be contributors to human obesity.

Characterization of the human dopamine D3 receptor expressed in transfected cell lines

A full-length cDNA clone of the human dopamine D3 receptor was obtained by the polymerase chain reaction (PCR) using reverse-transcribed RNA from human brain as the template. The cDNA was inserted into an expression vector which was then stably transfected into either Chinese hamster ovary (CHO), SK-N-MC human epithelioma or mouse CCL1.3 fibroblast cell lines. Post-transfection, the Bmax for D3 receptor expression was 1.9, 1.1 and 0.4 pmol/mg protein in the CHO-K1, SK-N-MC and CCL1.3 cell lines, respectively. The D3 receptor expressed in CHO-K1 and CCL1.3 cells exhibited similar radioligand binding profiles, especially for the D3-selective compound, 7-hydroxy-2-(di-n-propylamino)tetralin (7-OH-DPAT). Radioligand-binding competition curves of presumed D3 agonists were shifted to the right by the addition of guanine nucleotides and Na+ to the assay buffer. Presumed D3-receptor agonists had no effect on cAMP accumulation in any of the D3-transfected cell lines although cAMP accumulation was inhibited by dopamine D2 receptor activation in D2-transfected CHO and CCL1.3 cells and by activation of the exogenously expressed neuropeptide Y receptor in SK-N-MC cells. Also, D3 receptor activation neither potentiated ATP-stimulated arachidonic acid release from CHO cells nor stimulated inositol phosphate production in CCL1.3-cells although both of these responses were elicited by D2 agonists in D2-transfected cells. We conclude that the signalling properties of the D3 receptor differ from those of its closest homolog, the D2 receptor.

Differential interaction of β1- and β3-adrenergic receptors with Gi in rat adipocytes

Abstract The interaction of β1- and β3-adrenergic receptors and Gi proteins was examined in rat adipocytes. In intact adipocytes, cyclic AMP accumulation stimulated by the β3-selective agonist, BRL 37344 (BRL), was potentiated by pertussis toxin (PTX), as was the β1-sensitive component of isoproterenol (ISO)-stimulated cyclic AMP accumulation. These data suggest that β1- and β3-receptors interact with both Gs and Gi in intact adipocytes. F analysis of the activation of adenylyl cyclase by the β-receptor subtypes was performed in adipocyte membranes in which the activity of Gi was manipulated by both GTP and PTX. Unlike cyclic AMP accumulation in cells, the activation of membrane adenylyl cyclase by ISO could be clearly resolved into components mediated by β1-(high affinity) to 0.1 μM GTP, but the activity mediated by β1-receptors was not. As a consequence, the proportion of total ISO-stimulated activity that was mediated by β3-receptors was significantly reduced at concentrations of GTP in which Gi proteins are active. Adenylyl cyclase activity stimulated by BRL was also inhibited at high concentrations of GTP. PTX abolished the inhibition of β3-receptor-stimulated activity by high GTP concentrations. This is the first study to indicate that Gi proteins can limit β3- but not β1-stimulated adenylyl cyclase activity and are consistent the hypothesis that β3-receptors interact with both Gs and Gi, whereas β1-receptors couple predominantly to

Discovery of selective dopamine D4 receptor antagonists: 1-Aryloxy-3-(4-aryloxypiperidinyl)-2-propanols

Abstract High volume screening identified 3-(4-benzylpiperidinyl)-1-naphthoxy-2-propanol as a selective dopamine D4 receptor ligand. A systematic structure-activity study revealed that the benzyl group could be replaced with phenoxy and the naphthalene with phenyl to improve potency almost tenfold. The (R) enantiomer of this compound had a D4 affinity of 2 nM and was over 100-fold weaker at dopamine D2 and D3 receptors.

4-bromo-1-methoxy-N-[2-(4-aryl-1-piperazinyl)ethyl]-2-naphthalenecarboxamides: Selective dopamine D3 receptor partial agonists

Abstract A series of 4-bromo-1-methoxy-N-[2-(4-aryl-1-piperazinyl)ethyl]-2-naphthalenecarboxamide dopamine (DA) D 3 receptor agonists has been identified. These compounds were found to be selective for DA D 3 over D 2 receptors and were shown to be partial to full agonists as measured by stimulation of mitogenesis in D 3 -transfected CHO p-5 cells.

8-amino-6-(arylsulphonyl)-5-nitroquinolines: novel nonpeptide neuropeptide Y1 receptor antagonists

A novel series of 8-amino-6-(arylsulphonyl)-5-nitroquinoline neuropeptide Y1 (NPY) receptor antagonists is reported. The 8-amino and 5-nitro groups were important for NPY1 binding affinity as changes caused large drops in potency. The 6-arylsulphonyl group was necessary; however, substitution on the phenyl was tolerated. The 2-isopropyl analog 21 was a moderately potent, highly selective NPY1 receptor antagonist.

Expression and characterisation of human and rat CRF2α receptors

Rat and human CRF2alpha receptors were expressed in CHO-pro5 cells and stable cell lines generated. Each receptor was characterised using [125I][tyr0]sauvagine and results compared to CRF1 receptors expressed in the same parental cell line. Under identical assay conditions, [125I][tyr0]sauvagine labelled both CRF1 and CRF2alpha receptors with high affinity. The level of expression varied from 103 fmol/mg membrane protein to 1842 fmol/mg membrane protein (rat CRF1 receptors and rat CRF2 receptors, respectively). It was possible to establish robust scintillation proximity assays (SPA) using wheat germ agglutinin (WGA) SPA beads to trap membrane protein. The success of the SPA assay format was dependent on the level of receptor expression observed. The rank order of affinities of a series of peptide CRF receptor agonists and antagonists was similar to that described in the literature for the two receptor subtypes as determined using radioligand binding and cAMP accumulation. No pharmacological differences were apparent between rat and human cloned receptors with the exception of alpha-helical CRF-(9-41). This peptide exhibited 10-fold higher affinity for rat CRF2alpha receptors as compared to human CRF2alpha receptors. PD 173307, PD 173602 and PD 174239 exhibited high affinity and selectivity for human CRF1 receptors, and as such represent useful tools for probing CRF receptor function.

Discovery of selective dopamine D3 ligands: I. Dimeric 2-[4-(3-aminopropoxy)phenyl]benzimidazole antagonists

Abstract A novel series of dimeric 2-[4-(3-aminopropoxy)phenyl]benzimidazole dopamine (DA) D3 receptor antagonists has been discovered. Most of the dimeric structure is needed for DA binding activity; however, a second basic nitrogen atom is not required. A representative compound had no effects on DA synthesis in rat brain but inhibited spontaneous locomotor activity in mice and stimulated locomotor activity in habituated rats.

Discovery of selective dopamine D3 ligands: II. 2-[4-[3-(4-aryl-1-piperazinyl)propoxy]phenyl]benzimidazole partial agonists

Abstract A novel series of 2-[4-[3-(4-aryl-1-piperazinyl)propoxy]phenyl]benzimidazole dopamine D3 receptor agonists has been discovered. The aryl group was crucial for activity and Topliss analysis confirmed that phenyl was optimal for DA D3 receptor binding and selectivity. The phenyl analogue 3 was a partial agonist in a second messenger assay. It increased DA synthesis in rat brain and inhibited exploratory locomotor activity in rodents.

Substitution of D-Trp32 in NPY Destabilizes the Binding Transition State to the Y1 Receptor Site in SK-N-MC Cell Membranes

The retention rate of the spin label 3-isothiocyanto methyl-2,2,5,5-tetramethyl-1-pyrrolidinyl oxyl spin label (proxyl) attached to the porcine N-acetyl-NPY peptide and the porcine N-acetyl-D-Trp32-NPY peptide at Lys4 was investigated using SK-N-MC neuroblastoma cell membranes containing the Y1 receptor. The release rate of the spin labeled peptides was monitored by electron spin resonance and the KD was determined by a direct radiolabeled NPY displacement binding assay. The analyses show that for the porcine [Ac-Tyr1Nε4-proxyl]-NPY, the KD was 8 × 10−10 M and koff was 2.7 × 10−4 sec−1 yielding a value for kon of 3.3 × 105 sec−1 M−1. The [Ac-Tyr1, Nε4-proxyl,-D-Trp32]-NPY antagonist ligand had a value of KD equal to 1.35 × 10−7 M and koff was 1.7 × 10−4 sec−1 leading to a value for kon of 1.2 × 103 sec−1 M−1. The difference in the kon rates of two orders of magnitude is interpreted as demonstrating the N-acetyl-Nε4 proxyl-D-Trp32-NPY ligand binding transition state to be of higher energy then for the unmodified NPY amino acid sequence.