Heterotrimeric guanine nucleotide–binding protein (G protein)–coupled receptors constitute the largest family of eukaryotic signal transduction proteins that communicate across the membrane. We report the crystal structure of a human β2-adrenergic receptor–T4 lysozyme fusion protein bound to the partial inverse agonist carazolol at 2.4 angstrom resolution. The structure provides a high-resolution view of a human G protein–coupled receptor bound to a diffusible ligand. Ligand-binding site accessibility is enabled by the second extracellular loop, which is held out of the binding cavity by a pair of closely spaced disulfide bridges and a short helical segment within the loop. Cholesterol, a necessary component for crystallization, mediates an intriguing parallel association of receptor molecules in the crystal lattice. Although the location of carazolol in the β2-adrenergic receptor is very similar to that of retinal in rhodopsin, structural differences in the ligand-binding site and other regions highlight the challenges in using rhodopsin as a template model for this large receptor family.

/pdf/high-resolution-crystal-structure-of-an-engineered-human-b2-228wsbm59h.pdf

High-Resolution Crystal Structure of an Engineered Human β2-Adrenergic G Protein–Coupled Receptor

http://www.back-in-business-physiotherapy.com/images/files/DescendingControl.pdf

Descending control of pain.

Stem cells are defined as cells able to both extensively self-renew and differentiate into progenitors. Research data show that more resistant stem cells than common cancer cells exist in cancer patients.To identify unrecognized differences between cancer stem cells and cancer cells might be able to develope effective classification,diagnose and treat ment for cancer.

Stem Cells,Cancer and Cancer Stem Cells

Cutaneous melanin pigment plays a critical role in camouflage, mimicry, social communication, and protection against harmful effects of solar radiation. Melanogenesis is under complex regulatory control by multiple agents interacting via pathways activated by receptor-dependent and -independent mechanisms, in hormonal, auto-, para-, or intracrine fashion. Because of the multidirectional nature and heterogeneous character of the melanogenesis modifying agents, its controlling factors are not organized into simple linear sequences, but they interphase instead in a multidimensional network, with extensive functional overlapping with connections arranged both in series and in parallel. The most important positive regulator of melanogenesis is the MC1 receptor with its ligands melanocortins and ACTH, whereas among the negative regulators agouti protein stands out, determining intensity of melanogenesis and also the type of melanin synthesized. Within the context of the skin as a stress organ, melanogenic activity serves as a unique molecular sensor and transducer of noxious signals and as regulator of local homeostasis. In keeping with these multiple roles, melanogenesis is controlled by a highly structured system, active since early embryogenesis and capable of superselective functional regulation that may reach down to the cellular level represented by single melanocytes. Indeed, the significance of melanogenesis extends beyond the mere assignment of a color trait.

https://www.physiology.org/doi/pdf/10.1152/physrev.00044.2003

Melanin Pigmentation in Mammalian Skin and Its Hormonal Regulation

G Protein-coupled Receptors I. DIVERSITY OF RECEPTOR-LIGAND INTERACTIONS

The alpha 1-adrenergic receptors activate a phospholipase C enzyme by coupling to members of the large molecular size (approximately 74 to 80 kilodaltons) G alpha h family of guanosine triphosphate (GTP)-binding proteins. Rat liver G alpha h is now shown to be a tissue transglutaminase type II (TGase II). The transglutaminase activity of rat liver TGase II expressed in COS-1 cells was inhibited by the nonhydrolyzable GTP analog guanosine 59-O-(3-thiotriphosphate) or by alpha 1-adrenergic receptor activation. Rat liver TGase II also mediated alpha 1-adrenergic receptor stimulation of phospholipase C activity. Thus, G alpha h represents a new class of GTP-binding proteins that participate in receptor signaling and may be a component of a complex regulatory network in which receptor-stimulated GTP binding switches the function of G alpha h from transglutamination to receptor signaling.

http://science.sciencemag.org/content/sci/264/5165/1593.full.pdf

Gh: A GTP-binding protein with transglutaminase activity and receptor signaling function

The α1ARs are important mediators of sympathetic nervous system responses, particularly those involved in cardiovascular homeostasis, such as arteriolar smooth muscle constriction and cardiac contraction.1 2 In addition, α1ARs have more recently been implicated in the pathogenesis of cardiac hypertrophy, in ischemia-induced cardiac arrhythmias, and in ischemic preconditioning.1 3 Like other ARs, α1ARs are activated by the catecholamines, norepinephrine and epinephrine. They are intrinsic membrane glycoproteins and are members of the GPCR superfamily.

Over the past 10 to 15 years, data initially based on functional, radioligand, and biochemical studies have accumulated, indicating that the α1ARs are a heterogeneous group of distinct but related proteins. This conclusion has been confirmed with the molecular cloning of three distinct α1-receptor subtypes, although until recently discrepancies between the properties of the cloned expressed receptors and those characterized pharmacologically and biochemically have led to confusion in the classification of α1-receptor subtypes and their coupled effector responses.

As detailed in the present review, much of this confusion has now been clarified for the three cloned α1ARs. These and other recent insights into the molecular structure, function, and signaling of α1ARs, the control of α1AR-gene expression, and pharmacological evidence for additional α1AR subtypes will be reviewed here. For additional information, the reader is also referred to several previous reviews of α1ARs.4 5 6 7 

Functional studies of AR responses, particularly from the laboratories of McGrath8 and Ruffolo,9 provided the initial evidence that there may be subtypes of α1ARs. These studies indicated that postjunctional responses mediated by α1ARs could not be explained adequately on the basis of a single population of receptors. This concept was further advanced …

α1-Adrenergic Receptor Subtypes Molecular Structure, Function, and Signaling

alpha 1-Adrenergic receptor (alpha 1-AR) subtypes (alpha 1A and alpha 1B) play a critical role in vascular smooth muscle contraction and circulatory homeostasis. Transcripts for these guanine nucleotide-binding protein-coupled receptors are extremely low in abundance, however, and isolation of their cDNAs is difficult. We have developed a novel technique for identifying rare clones in a cDNA library, which has been used successfully to isolate a cDNA clone encoding an alpha 1D-AR. A 564-bp polymerase chain reaction product encoding a region between the third and sixth transmembrane domains of the alpha 1D-AR was first generated using rat brain mRNA as template and highly degenerate primers. The primers corresponded to those domains but contained mismatches to the alpha 1B-AR sequences. A 3-kb transcript was identified with this polymerase chain reaction probe, by Northern analysis of rat hippocampus. However, traditional plaque hybridization failed to identify a cDNA in a rat hippocampus lambda gt10 library. By solution-phase screening of virtually the entire library, a cDNA containing a 3-kb insert was identified, amplified, and purified. This insert encodes a 560-amino acid protein corresponding to the topology of guanine nucleotide-binding protein-coupled receptors. This receptor has approximately 71% amino acid identity, in the transmembrane regions, to the hamster and rat alpha 1B-ARs. Characterization of the receptor expressed in COS-7 cells, by ligand binding and photoaffinity labeling, revealed some of the characteristics of an alpha 1A-AR. However, unlike alpha 1A-ARs characterized previously in membrane preparations or in solubilized partially purified preparations, the expressed receptor could be extensively inactivated by chlorethylclonidine. In addition, it displays ligand-binding properties that are not consistent with an alpha 1A-AR. This indicates that the cDNA clone that we have isolated encodes a novel alpha 1-AR subtype, which we classify as the alpha 1D-AR.

Solution-phase library screening for the identification of rare clones: isolation of an alpha 1D-adrenergic receptor cDNA.

The adrenergic receptors play a key role in the modulation of sympathetic nervous system activity as well as a site of action for many therapeutic agents. The alpha1-adrenergic receptor subtypes (alpha1A-, alpha1B-, alpha1D) are the prime mediators of smooth muscle contraction and hypertrophic growth, but their characterization in both binding and function have lagged the other adrenergic family members. Although they are derived from a related ancestral gene and all nine adrenergic receptor family members bind the endogenous ligands, epinephrine and norepinephrine, with roughly similar affinities, there are major differences in the mode of binding, second messenger utilization, and physiological effects of the alpha1-subtypes compared with beta- or alpha2-subtypes. Here, we review the recent literature on aspects of its binding pocket and how it differs from the beta-adrenergic receptor paradigms. We also review the signaling components and aspects of its function and provide new insights into its roles in smooth muscle, growth, neurological, and cardiovascular function.

Alpha1-adrenergic receptors: new insights and directions

Studies have been amassed in the past several years indicating that an agonist can conform a receptor into an activation state that is dependent upon an intrinsic property of the agonist usually based upon its chemical composition. Theoretically, each different agonist could impart its own unique activation state. Evidence for multiple signaling states for the G-protein-coupled receptors will be reviewed and is derived from many different pharmacological behaviors: efficacy, kinetics, protean agonism, differential desensitization and internalization, inverse agonism, and fusion chimeras. A recent extension of the ternary complex model is suggested by evidence that the different processes that govern deactivation, such as desensitization and internalization, is also regulated by conformers specific to the agonist. Rhodopsin may serve as a primer for the study of multiple activation states. Therapeutic implications that utilize multiple signaling states hold vast promise in the rationale design of drugs.

Dianne M. Perez

Papers

Gh: A GTP-binding protein with transglutaminase activity and receptor signaling function

α1-Adrenergic Receptor Subtypes Molecular Structure, Function, and Signaling

Solution-phase library screening for the identification of rare clones: isolation of an alpha 1D-adrenergic receptor cDNA.

Alpha1-adrenergic receptors: new insights and directions

Multiple Signaling States of G-Protein-Coupled Receptors