Daniel K. Rohrer

A monoclonal antibody for G protein–coupled receptor crystallography

G protein–coupled receptors (GPCRs) constitute the largest family of signaling proteins in mammals, mediating responses to hormones, neurotransmitters, and senses of sight, smell and taste. Mechanistic insight into GPCR signal transduction is limited by a paucity of high-resolution structural information. We describe the generation of a monoclonal antibody that recognizes the third intracellular loop (IL3) of the native human β2 adrenergic (β2AR) receptor; this antibody was critical for acquiring diffraction-quality crystals.

Alterations in dynamic heart rate control in the β1-adrenergic receptor knockout mouse

beta 1-Adrenergic receptors (beta 1-ARs) are key targets of sympathetic nervous system activity and play a major role in the beat-to-beat regulation of cardiac chronotropy and inotropy. We employed a beta 1-AR gene knockout model to test the hypothesis that beta 1-AR function is critical for maintenance of resting heart rate and baroreflex responsiveness and, on the basis of its important role in regulating chronotropy and inotropy, is also required for maximal exercise capacity. Using an awake unrestrained mouse model, we demonstrate that resting heart rate and blood pressure are normal in beta 1-AR knockouts and that the qualitative responses to baroreflex stimulation are intact. Chronotropic reserve in beta 1-AR knockouts is markedly limited, with peak heart rates approximately 200 beats/min less than wild types. During graded treadmill exercise, heart rate is significantly depressed in beta 1-AR knockouts at all work loads, but despite this limitation, there are no reductions in maximal exercise capacity or metabolic indexes. Thus, in mice, the beta 1-AR is not essential for either maintenance of resting heart rate or for maximally stressed cardiovascular performance.β1-Adrenergic receptors (β1-ARs) are key targets of sympathetic nervous system activity and play a major role in the beat-to-beat regulation of cardiac chronotropy and inotropy. We employed a β1-AR gene knockout model to test the hypothesis that β1-AR function is critical for maintenance of resting heart rate and baroreflex responsiveness and, on the basis of its important role in regulating chronotropy and inotropy, is also required for maximal exercise capacity. Using an awake unrestrained mouse model, we demonstrate that resting heart rate and blood pressure are normal in β1-AR knockouts and that the qualitative responses to baroreflex stimulation are intact. Chronotropic reserve in β1-AR knockouts is markedly limited, with peak heart rates ∼200 beats/min less than wild types. During graded treadmill exercise, heart rate is significantly depressed in β1-AR knockouts at all work loads, but despite this limitation, there are no reductions in maximal exercise capacity or metabolic indexes. Thus, in mice, the β1-AR is not essential for either maintenance of resting heart rate or for maximally stressed cardiovascular performance.

The developmental and physiological consequences of disrupting genes encoding beta 1 and beta 2 adrenoceptors.

Publisher Summary The role that individual β AR subtypes play in specific physiological processes has been traditionally defined using subtype-specific ligands. Unfortunately, many of these ligands either possess poor selectivity or are used at concentrations in vivo that can lead to occupation of nonspecific subtypes. The advent of gene disruption techniques in the mouse now enables to selectively delete or alter cloned genes as a means of identifying the specific function(s) of their gene products. To better understand β AR subtype-specific functions in the context of either the whole animal or isolated organs and cells, the genes encoding both β 1 - and β 2 ARs have been disrupted. Gene targeting vectors containing β 1 AR or β 2 AR gene sequences interrupted or partially replaced by a bacterial neomycin resistance gene cassette were flanked by a viral thymidine kinase gene cassette. A standard positive-negative selection strategy (G418 + gancyclovir) has been used to isolate R1 embryonic stem (ES) cells, having undergone homologous recombination at β 1 AR or β 2 AR loci. The pharmacological profile of β 1 AR and β 2 AR knockouts is consistent with the loss of these specific receptor subtypes. When the nonspecific β AR antagonist [ 125 I]cyanopindolol is used in competition binding studies, excess unlabeled CGP 20712A ( β 1 AR-specific antagonist) reveals a selective loss of specific high-affinity sites in β 1 AR knockout heart or lung membranes while excess unlabeled ICI 118,551 ( β 2 AR-specific antagonist) reveals a selective loss of specific high-affinity sites in β 2 AR knockout lung membranes. These results provide further evidence that the β 1 AR and β 2 AR gene disruptions have abolished β AR protein expression. The physiological impact of knocking out either β 1 - or β 2 ARs has been studied in instrumented animals and in isolated tissues.

Crystal Structures of the β2-Adrenergic Receptor

G protein coupled receptors (GPCRs) constitute the largest family of membrane proteins in the human genome, and are responsible for the majority of signal transduction events involving hormones and neuro-transmitters across the cell membrane. GPCRs that bind to diffusible ligands have low natural abundance, are relatively unstable in detergents, and display basal G protein activation even in the absence of ligands. To overcome these problems two approaches were taken to obtain crystal structures of the β2-adrenergic receptor (β2AR), a well-characterized GPCR that binds cate-cholamine hormones. The receptor was bound to the partial inverse agonist carazolol and co-crystallized with a Fab made to a three-dimensional epitope formed by the third intracellular loop (ICL3), or by replacement of ICL3 with T4 lysozyme. Small crystals were obtained in lipid bicelles (β2AR-Fab) or lipidic cubic phase (β2AR-T4 lysozyme), and diffraction data were obtained using microfocus technology. The structures provide insights into the basal activity of the receptor, the structural features that enable binding of diffusible ligands, and the coupling between ligand binding and G-protein activation.

Cardiovascular β-Adrenergic Receptor Subtype Physiology Studied by Targeted Gene Disruption • 95

Cardiovascular β-Adrenergic Receptor Subtype Physiology Studied by Targeted Gene Disruption • 95

Defective myogenesis in NFB-s mutant associated with a saturable suppression of MYF5 activity.

Myogenic cell lines have proved to be useful tools for investigating the molecular mechanisms that control cellular differentiation. NFB-s is a mutant myogenic cell line which fails to differentiate in vitro, and can repress differentiation in normal myogenic cells when fused to form heterokaryons. The NFB-s cell line was used here to study the molecular mechanisms underlying such myogenic repression. Using muscle-specific reporter genes, we show that NFB-s cells fail to activate fully the muscle differentiation program at a transcriptional level, although muscle-specific transcription can be enhanced by regulators of differentiation such as pertussis toxin. Paradoxically, we find that the myogenic regulator myf5 is expressed at constitutively high levels in NFB-s cells, and retains DNA binding activity. Expression plasmids encoding NFB-derived myf5 cDNA can rescue the myogenic phenotype in NFB-s cells, demonstrating that a threshold level of positive regulators must be reached before the myogenic program is activated. Thus, the dominant negative phenotype does not appear to result from defective myf5, but is due to a dosage-dependent saturable mechanism that inteferes with myf5 function. These studies demonstrate that the stoichiometric ratio of positive and negative regulators is critical for determining the myogenic differentiation state.