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At present, the only therapeutic agent in current use that is known to exert its effects through the GABA-B receptor is baclofen, the β-chlorophenyl analog of GABA. This compound is used extensively as a centrally active muscle relaxant, but it has been shown clinically to exert additional effects, including analgesia, suppression of drug addiction, and cessation of chronic cough. Basic research indicates that there may be many other applications not only for agonists, but also antagonists at the GABA-B receptor, and this chapter seeks to examine the potential significance of these and also of allosteric modulators for the receptor, which have been recognized during the past 5 yr. GABA-B receptors have been implicated in neuronal processing in many brain regions and there is considerable evidence for their pathological involvement in various diseases of the central nervous system, such as absence epilepsy, depression, and even in the etiology of nicotine dependency in humans. Novel chemical entities will surely be the key to exploiting this receptor. At present, the only agonist (baclofen) in the clinic frequently produces unwanted side effects, and this clearly limits its usage. There are no antagonists in clinical therapy, although trials for cognition deficits are underway. Metabotropic receptors, of which the GABA-B receptor is a member, provide most of the targets for our currently used therapeutic agents; thus, there seems to be every reason to believe that the rewards for discovering clinically available GABA-B receptor ligands could be high. [ABSTRACT FROM AUTHOR]

γ-Aminobutyric acid B (GABAB) receptors are the G protein-coupled receptors for the inhibitory neurotransmitter GABA. Cloning efforts revealed that functional GABAB receptors can be assembled from the products of the GABAB1 and GABAB2 genes. However, cloned receptors do not exhibit the reported functional and pharmacological diversity of native receptors, which led to speculations about the existence of additional GABAB receptor genes. Several groups now have generated mice with ablations in the GABAB1 and GABAB2 genes, which allowed studying the contributions of cloned gene products to pre- and postsynaptic GABAB functions. In this chapter, we review the phenotypes of GABAB knockout mice and discuss how genetic experiments have contributed to our current understanding of the GABAB receptor system. [ABSTRACT FROM AUTHOR]

Functional γ-aminobutyric acid (GABAB) receptors are formed by the association of GABABR1 and GABABR2, two closely related subunits belonging to the G protein coupled receptor superfamily. An increasing number of reports have now uncovered that GABAB receptors display unique functional properties, including unusual activation and inactivation mechanisms following binding of agonists. In this chapter the focus will be on nonclassical aspects of GABAB receptor phosphorylation, desensitization, downregulation, signaling, and association with a variety of protein partners. In addition, the extent to which the heterodimeric structure of the receptor contributes to establish these unusual properties will be discussed. [ABSTRACT FROM AUTHOR]

Nearly 10 yr have passed since the authors review "Chemistry of GABAB Modulators" appeared in the book "The GABA Receptors," 2nd edition, edited by S. J. Enna and N. G. Bowery, Humana Press, Totowa, 1997 (1). In this update the authors wish to outline only new developments not covered in the 1997 paper. Baclofen, synthesized for the first time in September 1962, is still the only γ-aminobutyric acid (GABAB)-receptor agonist marketed for the treatment of spasticity and trigeminal neuralgia. It is fascinating to learn how many highly competent chemists devised manifold and elegant synthetic procedures for either racemic or (R)-(−)-baclofen and the structurally closely related potent antidepressant (R)-(−)-Rolipram, a selective phosphodiesterase-4 inhibitor. The new syntheses published after 1996 are listed in alphabetical order (2-16). Very recently a prodrug of (R)-(−)-baclofen (Fig. 1) was described which enhanced the oral bioavailability in Cynomolgus monkeys to more than 80% (17). It is planned to test this prodrug in clinical trials for the treatment of spasticity and gastroesophageal reflux disease. [ABSTRACT FROM AUTHOR]

The γ-aminobutyric acid (GABA) neurotransmitter acting through ionotropic and metabotropic receptor classes exerts the major inhibitory control in the central nervous system. Therapeutic agents targeting GABA receptors (GABA-R), such as benzodiazepines and baclofen, are used to treat many nervous system conditions, including anxiety and spasticity. The subunit composition of GABA-Rs at the cell surface plays a critical role in determining their physiological and pharmacological properties, and alteration of GABA-R subunit expression has been associated with a number of diseases including schizophrenia, temporal lobe epilepsy, and alcoholism. The ionotropic type A GABA receptor (GABAAR) and type C GABA receptor (GABACR) are pentameric complexes that comprise a ligand gated chloride channel. The metabotropic type B GABA receptor (GABABR) is a heterodimer that couples G protein-signaling to GABA binding. There are eight classes of ionotropic receptor subunits and only two metabotropic receptor subunit classes. Most of the GABAAR subunit genes are localized in syntenic β-α-γ gene clusters on four chromosomes but the two GABABR genes are localized on distinct chromosomes. Control over subunit expression in different brain regions and during development is orchestrated at the genomic level by the use of multiple promoter regions and through the alternative splicing of GABA-R subunit RNAs. This chapter examines current GABA-R research relevant to the many levels of control over receptor gene regulation and cell surface receptor expression that may be relevant to both health and disease. [ABSTRACT FROM AUTHOR]

Ligands that act at γ-aminobutyric type A (GABAA) receptors, in particular the benzodiazepines and related drugs, have broad clinical use but also the liability for abuse and dependence. Recent epidemiological data suggest that abuse of benzodiazepine-type drugs may be on the upswing, with a shift from primary misuse of benzodiazepines by people in a therapeutic setting to use by younger people engaging in recreational abuse. Laboratory findings suggest that benzodiazepine-type drugs have reinforcing effects both in human and non-human subjects. However, benzodiazepine-type drugs appear to have lower reinforcing effectiveness compared to other drugs of abuse, such as psychomotor stimulants. Recent research has begun to explore the role of GABAA receptor subtypes in the reinforcing effects of benzodiazepine-type drugs, and unlike other behavioral effects (e.g., motor coordination deficits); reinforcing effects are not easily attributed to a single receptor subtype. Perhaps the most firm conclusion that can be made at this point is that stimulation of GABAA receptors containing α1 subunits (α1GABAA receptors) is not necessary for self-administration of benzodiazepine-type compounds. Benzodiazepine use also is associated with physical dependence, characterized by a withdrawal syndrome. In both human and non-human subjects, this withdrawal syndrome is considered to be intermediate in severity. Preliminary results suggest that compounds with selectivity for α2GABAA, α3GABAA, and/or α5GABAA receptors do not induce physical dependence. As with reinforcing effects, systematic studies with selective compounds having relatively high intrinsic efficacy at particular subtypes should shed light on theses important mechanistic issues. [ABSTRACT FROM AUTHOR]

Idiopathic generalized epilepsies (IGEs) are characterized by absence, myoclonic, and/or primary generalized tonic-clonic seizures in the absence of structural brain abnormalities and are believed to have a genetic basis. Mutations in γ-aminobutyric acid (GABAA) receptor γ2-, α1-, and δ-subunits have been associated with different IGE syndromes, and all of the γ2-subunit mutations are also associated with febrile seizures. Here, mutations in GABAA receptor γ2-, α1-, and δ-subunits that have been associated with different IGE syndromes will be reviewed and the basis for the defect in GABAergic inhibition associated with febrile seizures will be explored. These mutations have been shown to alter GABAA receptor gating, expression, and/or trafficking of the receptor to the cell surface, all pathophysiological mechanisms that result in reduced GABA-evoked currents that, in turn, would cause neuronal disinhibition in neurons and thus predispose affected patients to manifest afebrile and febrile seizures. [ABSTRACT FROM AUTHOR]

γ-Aminobutyric acid A (GABAA) receptor subunit multiplicity results in the generation of diverse receptor subtypes with distinct biophysical and pharmacological properties. These receptor subtypes show different subcellular targeting and can be activated by GABA in different ways, with different functional outcomes. Synaptic receptors underlie brief postsynaptic currents, whereas high-affinity, largely extrasynaptic, receptors generate a "tonic" conductance. These differences have implications for our understanding of normal brain function and the rational design of therapeutic agents. [ABSTRACT FROM AUTHOR]

γ-Aminobutyric acid (GABAA) receptors are the major inhibitory neurotransmitter receptors in the brain and are the site of action of many clinically important drugs. They are made up of five subunits that can belong to eight different subunit classes. Depending on their subunit composition these receptors exhibit distinct pharmacological and electrophysiological properties. The distinct but overlapping regional and cellular distribution of these subunits gives rise to the formation of a large number of GABAA-receptor subtypes, only a few of which so far have been unequivocally identified in the brain. Recent homology modeling of the structure of the extracellular and transmembrane domain of GABAA receptors for the first time provided a glimpse on the three-dimensional organization of this receptor, and placed different protein segments that have been shown to be of functional importance into a "region in space" and into defined neighboring relations. The models obtained could explain experimental observations and propose the location of putative drug-binding sites. They can be now used to design experiments for clarification of pharmacological and structural questions as well as to shed light on conformational changes during binding of agonist, gating, and allosteric modulation of these receptors. [ABSTRACT FROM AUTHOR]

γ-Aminobutyric acid receptors (GABAA Rs) are the principal receptors that mediate neural inhibition in the brain. Changes in the function of GABAergic transmission are implicated in activity-dependent adaptation of neural excitability. Of particular interest are mechanisms that control the size of the postsynaptic GABAA-receptor pool, a major determinant of synaptic strength. Mechanisms of trafficking of postsynaptic GABAA Rs contribute to regulation of inhibitory synaptic transmission in response to changes in neural activity and extracellular stimuli. This review summarizes current information available on the receptor structures relevant for trafficking of GABAA Rs, the molecular composition of the submembrane cytoskeleton of inhibitory synapses, and the receptor interacting proteins that regulate the localization and trafficking of postsynaptic GABAA Rs during exocytosis, lateral diffusion and endocytic recycling, and degradative pathways. [ABSTRACT FROM AUTHOR]

Oscillatory activities are hallmarks of neuronal network functions. They contribute to information processing in a multitude of brain activities including cognitive functions. A key requirement for the generation of network oscillations is regular and synchronized neuronal activity. The temporal structure and spatial coherence of network oscillations is largely controlled by GABAergic interneurons. To provide the appropriate response characteristics the synaptic specialization of these interneurons includes an extensive diversity of GABAA receptors. The cell- and domain-specific location of these receptor subtypes offers the possibility to gain functional insights into the role of defined neuronal circuits. Pharmacologically, neurons operating with α1 GABAA receptors mediate sedation while the small population of α2GABAA receptors largely mediates anxiolysis. Schizophrenia-related phenotypes can be ameliorated through α3GABAA receptors. Memory and learning are enhanced by diminishing α5GABAA receptor function. Thus, by targeting selective neuronal circuits GABAA receptor subtypes provide a new circuit-specific GABA pharmacology for a multitude of CNS dieases. [ABSTRACT FROM AUTHOR]

γ-Aminobutyric acid (GABA), an amino acid neurotransmitter, is widely distributed throughout the neuraxis. Two pharmacologically and molecularly distinct GABA receptors have been identified, GABAA and GABAB. GABAA receptors are pentameric ligand-gated chloride-ion channels, whereas GABAB receptors are heterodimeric G protein-coupled sites. Although GABAA receptor subtypes can display pharmacological differences, the two molecularly distinct GABAB receptors have similar substrate specificities, limiting the ability to selectively manipulate this site. Gene deletion and point mutation studies have revealed the importance of GABA receptors in neural development and function, with subtle modifications in subunit amino acid composition having profound effects on behavioral phenotype and responses to drugs. The characterization of GABAA receptors has contributed substantially to the knowledge about allosteric regulation of ligand-gated ion channels. Such information is invaluable in defining precisely the mechanisms of action of numerous drugs, such as the benzodiazepines, and toxic agents. Research on GABAB receptors has proven the existence of dimeric metabotropic receptors and has provided the chemical tools necessary for defining such systems. The characterization of the pentameric GABAA and dimeric GABAB receptors has been crucial for understanding the neurobiological basis of some nervous system disorders. Given the importance of GABA in central nervous system function, further work on its receptors is likely to yield novel therapeutics for treating a host of neurological and psychiatric conditions. [ABSTRACT FROM AUTHOR]

In this chapter, we will give a perspective of in vitro assays used in drug discovery when targeting chemokine receptors. We outline the complexity of the chemokine system and give a historical perspective on the in vitro assays and types of assays used at different stages of discovery, followed by several examples of successes and failures in clinical trials. Finally, we discuss the rationale for continuing, after several failures, to target chemokine receptors and how screening may change with the increasing evidence of dimerization of chemokine receptors. [ABSTRACT FROM AUTHOR]

Actions of chemokines and the interaction with specific receptors within the central nervous system (CNS) surpass their original defined role of leukocyte recruitment to inflamed tissues. Chemokine receptor expression by resident CNS cells is crucial for normal brain development and architectural organization, neuronal protection during inflammatory and neurotoxic challenges, and, among many others, protective mechanisms during inflammatory conditions such as multiple sclerosis. The chemokine/chemokine receptor systems involved in such significant functions include CXCR4/CXCL12, CXCR2/CXCL1, and CX3CR1/CX3CL1. In this chapter, we discuss how these receptors might contribute to modulate communication within the CNS and with peripheral elements, and we also suggest potential mechanisms of action of fractalkine and the translation of these into the understanding of microglial function during neuro-inflammatory conditions. [ABSTRACT FROM AUTHOR]

Many chemokine receptors have now been detected in a variety of malignancies. Considerable data now exist that two receptors, CXCR4 and CCR7, are widely expressed in epithelial cancers and contribute to the ability of some tumors to metastasize. Preclinical studies support a role for these receptors in mediating tumor cell migration, but other functions have also been identified including support of tumor cell proliferation in response to ligand stimulation. Less is known about the function of other chemokine receptors expressed in malignant cells, but it is likely that at least some of these also contribute to aggressive behavior. Although the specific role of each receptor is complex, it is becoming clear that these receptors may mediate metastasis to some, but not all, metastatic sites characteristic of each histologic type. Some of the site specificity is due to expression of the cognate ligand at the secondary site; however, other factors to be defined also contribute to chemokine receptor-determined metastatic patterns. [ABSTRACT FROM AUTHOR]

Angiogenesis is the process of new blood vessel growth and is a critical biological process under both physiologic and pathologic conditions. Angiogenesis can occur under physiologic conditions that include embryogenesis and the ovarian/menstrual cycle. In contrast, pathologic angiogenesis is associated with chronic inflammation/chronic fibroproliferative disorders and tumorigenesis of cancer. Similarly, aberrant angiogenesis associated with chronic inflammation/fibroproliferative disorders is analogous to neovascularization of tumorigenesis of cancer. Net angiogenesis is determined by a balance in the expression of angiogenic compared with angiostatic factors. CXC chemokines are heparin-binding proteins that display unique disparate roles in the regulation of angiogenesis. Based on their structure, CXC chemokines can be divided into two groups that either promote or inhibit angiogenesis, and they are therefore uniquely placed to regulate net angiogenesis in both physiologic and pathologic conditions. [ABSTRACT FROM AUTHOR]

Tissue fibrosis, which results in the destruction of normal organ function, is a leading cause of morbidity and mortality. Current strategies for treating fibrosis have been unsuccessful, largely because of the difficulty in distinguishing whether inflammatory or fibrogenic events sustain the progression of the disease. The causes of fibrosis are diverse regardless of the tissue involved, and the common features include the sequential recruitment of inflammatory cells, overproliferation of matrix-producing cells, and the overproduction of extracellular matrix. An excessive wound-healing response presumably represents disruption in this sequence thereby leading to a disturbance in the balance between tissue remodeling, matrix degradation, and permanent scarring. The mechanisms involved in pulmonary fibrosis also represent three sequential events characterized by an initial insult, inflammation, and tissue repair. Central to the progression of these events is the balance between a T helper 1 (Th1) and a T helper 2 (Th2) environment, in which Th2-specific signals have been shown to be immunomodulatory and profibrotic. However, the release of these Th2-specific cytokines and chemokines by both inflammatory and resident cells maintains the fibrotic response, consequently leading to fibrotic disease. Evidence from animal models and human studies have identified a number of Th1/Th2-associated chemokines and chemokine receptors as profibrotic or antifibrotic. Therefore, investigating the chemokines, chemokine receptors, and the cells that they impact is an attractive approach to identifying therapeutic targets in fibrosis. [ABSTRACT FROM AUTHOR]

There have been tremendous advances made toward our understanding of chemokine receptor biology over the past decade. Much of the research conducted in this area was fueled by discoveries that certain chemokine receptor ligands (chemokines) could specifically block human immunodeficiency virus type 1 (HIV-1) infection and that certain chemokine receptors were the long-sought coreceptors that, together with CD4, were required for the productive entry of HIV-1, HIV-2, and simian immuno-deficiency virus (SIV) isolates. In the current survey, along with the interactions of the HIV-1 envelope glycoprotein with chemokine receptors, we have focused on the relationship certain chemokine receptors have with particular cellular systems such as lipid rafts, chemokine receptor-mediated signaling, and the actin cytoskeleton; all of which appear to play roles in or influence the establishment of HIV-1 infection. We will also discuss the dichotomous effects that chemokines display at both entry and postentry levels of HIV-1 infection and their potential significance for HIV-1 pathogenesis and acquired immunodeficiency syndrome (AIDS). [ABSTRACT FROM AUTHOR]

This chapter is an attempt to integrate recent studies concerning the role of chemokine receptors in the initiation, development, and maintenance of allergic lung diseases collectively referred to as asthma. The pathogenesis of asthma involves the coordinated trafficking of inflammatory cells to the lungs and draining lymph nodes, as well as the activation of these inflammatory cells. Chemokine receptors and their ligands play a prominent role in directing the inflammation associated with allergic lung disease. T lymphocyte-mediated immune responses can be broadly categorized as being type 1 or type 2, based on the cell types present and the associated cytokines produced. Allergic lung disease is a predominately type 2-mediated disease. The chemokine receptors CCR4, CCR6, and CCR8 serve to promote the recruitment of type 2 T (T helper 2; Th2) cells, whereas CXCR3 antagonizes type 2 and promotes type 1 T (T helper 1; Th1) cells. The pathophysiologic manifestations of asthma, including excessive mucus production, eosinophilia, and airway hyperreactivity, are dependent upon the trafficking and activation of eosinophils, mast cells, and goblet cells. Roles for chemokine receptors, including CCR4, CCR2, and CXCR4, in the trafficking and activation of these cell types during allergic lung disease are discussed. Finally, the incidence of allergic lung disease is increasing, and the costs associated with it are substantial. Chemokine receptor expression and use by inflammatory cells during allergic lung disease makes chemokine receptors an attractive therapeutic target. Implications for drug development are discussed in the context of experimental results. [ABSTRACT FROM AUTHOR]

Atherosclerosis is an inflammatory process that is strongly affected by chemokines that regulate the trafficking of inflammatory cells. A large amount of data from in vitro studies and murine models suggest an important role for chemokines in atherosclerosis. In man, genetic studies have revealed that specific polymorphisms in chemokine and chemokine receptor genes are associated with atherosclerotic diseases including coronary artery disease and carotid artery occlusive disease. Specifically, there are sound data supporting roles for the following receptors and their ligands: CCR2 and MCP-1 (CCL2); CX3CR1 and fractalkine (CX3CL1); CCR1, CCR5, and RANTES (CCL5); CXCR2 and IL-8 (CXCL8); CXCR6 and CXCL16; and CXCR3 and its ligands MIG (CXCL9), IP-10 (CXCL10) and I-TAC (CXCL11). These chemokines and their receptors participate in vascular inflammation via T-cell and monocyte chemoattraction, adhesion of monocytes to the vessel wall, and vascular smooth muscle cell migration and proliferation. Chemokines and chemokine receptors are being studied as potential therapeutic targets for the prevention or retardation of atherosclerotic disease. Although many of these therapies are promising, there are also limitations to specific chemokine-targeted therapy. [ABSTRACT FROM AUTHOR]

Rheumatoid arthritis (RA) affects 1% of the U.S. population, or 3 million people. RA is a chronic inflammatory disease characterized by synovial inflammation and hyperplasia, neovascularization, and progressive destruction of cartilage and bone. The cause(s) of RA remains poorly defined, but it is thought that both genetic and environmental factors, including molecular mimicry and host responses against cross-reactive antigens, lead to the development of autoimmune responses involving the joints. The goal of this chapter is to provide an overview of the contribution of members of the chemokine system to the immunopathogenesis of RA. This is of high clinical relevance because of the burgeoning interest in the use of chemokine and chemokine-receptor blockers in the treatment of a variety of inflammatory, autoimmune, and infectious disorders, including RA. [ABSTRACT FROM AUTHOR]

Chemokines exert multiple actions in inflammation, wound healing, and differentiation. Because these processes underlie aspects of the pathophysiology of allograft rejection, chemokine biology has been a major focus of transplantation studies. Although diverse sets of chemokines can be detected in allografts after ischemic reperfusion injury and acute and chronic rejection, recent studies now suggest that relatively few chemokine receptors play central roles in the development of transplant rejection. The antagonism of select chemokine receptors causes a reduction in leukocyte infiltration, prolongs graft function, and can synergize with other immune-suppressive regimens. Chemokine-targeted therapy may represent an important approach in the treatment of allograft rejection. [ABSTRACT FROM AUTHOR]

Chemokines regulate the process of hematopoiesis by controlling trafficking, proliferation/survival, and differentiation of hematopoietic stem and progenitor cells. Unique expression of the chemokine receptor CXCR4 in combination with adhesion molecules [very late antigen (VLA)-4, lymphocyte function-associated antigen-1 (LFA-1), and VLA-5] on early hematopoietic stem and progenitor cells makes their homing to bone marrow and seeding the stem cell niche possible. In bone marrow, the CXCL12-CXCR4 chemokine axis promotes the survival and retention of hematopoietic stem and early progenitor cells. In certain conditions, hematopoietic stem and progenitor cells are released from bone marrow to the blood circulation, a process called mobilization. Many agents that mobilize marrow progenitor cells induce or activate certain proteases of neutrophils. This leads to degradation of CXCL12 and, therefore, weakens the chemotactic activity of bone marrow. Whereas CXCL12 plays a positive role in survival and proliferation of hematopoietic stem and progenitor cells, many other chemokines suppress these processes. During their differentiation into mature cells, hematopoietic progenitor cells upregulate cell lineage-specific chemokine receptors to migrate to appropriate tissue sites. This cell type-specific switch in chemokine receptors and adhesion molecules is important for tissue-specific migration of hematopoietic cells, a process important for their differentiation or effector function in the periphery. [ABSTRACT FROM AUTHOR]

The ordered movement of lymphocytes through and positioning within lymphoid organs and peripheral sites is controlled by adhesion molecules together with chemokines and their receptors. Chemokine-mediated lymphocyte migration is critical for establishing the architecture of lymphoid organs and many aspects of lymphocyte function, including lymphocyte development, activation, and effector activity. Of the 19 chemokine receptors described in humans, all have been reported to be expressed on lymphocytes, and the expression pattern of chemokine receptors can itself be used to define and characterize lymphocyte subsets. For example, by using chemokine receptors, memory T cells can be split into distinct populations, such as central and effector memory T cells; and T helper 1 (Th1) and T helper 2 (Th2) cells exhibit distinguishable patterns of chemokine receptor expression, which can be used to study Th1/Th2 differentiation. Beyond their physiologic roles, chemokine receptors on lymphocytes are exploited by pathogens, such as in the use of CCR5 and CXCR4 by HIV-1 as coreceptors for viral entry. In this chapter, we will focus on the roles of chemokine receptors in lymphocyte trafficking in lymphoid organs and peripheral tissues and how understanding the chemokine system has shed light on larger issues in lymphocyte biology. We will discuss the roles of chemokines and chemokine receptors during the life cycles of lymphocytes—from early development through the acquisition of memory and effector functions. [ABSTRACT FROM AUTHOR]

Dendritic cell (DC) networks dictate peripheral tolerance and immunity in lymph nodes (LNs). The type, timing, location, and interaction of LN-recruited DC subtypes are pivotal and regulated by chemokines. We propose a concept that any DC subtype including myeloid and plasmacytoid DCs (mDCs and pDCs) is fundamentally categorized by three stages depending on the function and anatomical position: naïve DC, primed DC, and effector DC. Naïve mDC precursors are recruited to inflamed tissues in response to CCR1 and CCR5 ligands to become primed mDCs, remobilized to draining LNs in response to CCR7 ligands, and activated to become effector mDCs to undergo antigen-presenting function. In contrast, pDC precursors directly migrate to LNs in a CXCR3-dependent manner. LN-recruited, primed pDCs are activated to become effector pDCs that produce large amounts of cytokines and chemokines. Concerted recruitment and adequate network formation of distinct effector DCs are pivotal to determine the type and efficacy of immune response. [ABSTRACT FROM AUTHOR]

Neutrophils are the most abundant leukocytes in circulation. They rapidly infiltrate sites of tissue injury and play a critical role in innate immune responses. In addition, they also contribute to the development of adaptive immune responses. Isolation of the human chemokine IL-8 and the cloning of its receptors CXCR1 and CXCR2, followed by the cloning of their orthologues or homologues in animals, have enabled researchers to elucidate the mechanisms of neutrophil trafficking during immune responses at a molecular level. Since then, there has been tremendous progress in understanding how the trafficking of neutrophils is regulated by the chemokine/chemokine receptor system under not only pathologic but also physiologic conditions. In this chapter, the roles of the chemokine receptors in regulating the trafficking of neutrophils are described. [ABSTRACT FROM AUTHOR]

The biochemical events that are elicited upon chemokine engagement have been a major focus of interest in many cell types responding to a plethora of different chemokines. We now appreciate that collectively, chemokines can couple to a wide range of biochemical signals including phosphoinositide lipid metabolism, elevation of intracellular calcium levels, and activation of a wide array of protein and lipid kinases as well as small GTPases. Chemokine signaling events are particularly well studied in T lymphocytes where the ordered directional migration of T lymphocytes is a key process in development, immune surveillance, and immune responses. These cells therefore offer a splendid model system in which to understand the array of signals activated by chemokines and their functional importance. One of the most robust biochemical signals elicited by chemokines is the activation of several members of the phosphoinositide 3-kinase (PI3K) family. In many cell systems, PI3Ks are known to contribute to several aspects of the migratory machinery including gradient sensing, signal amplification, actin reorganization and hence cell motility. This chapter will therefore focus on the role of PI3K-dependent signaling events in T-lymphocyte migration and points at which these events may integrate with other effectors and signaling cascades. [ABSTRACT FROM AUTHOR]

To date, all mammalian chemokine receptors and chemokine-binding proteins belong to the seven-transmembrane (7TM)-spanning family of receptors, which are typically, although not exclusively, coupled to G proteins (1). The large G protein-coupled receptor (GPCR) family comprises almost 1000 members in the human genome (2,3) accounting for in excess of 3% of transcribed sequences. This family is subdivided into the class A, B, C, and F/S families on the basis of shared motifs, and chemokine receptors specifically belong to the class A GPCR family. This review summarizes the current state of knowledge of chemokine receptor structure and discusses recent developments toward the determination of the full three-dimensional structure of these important receptors. [ABSTRACT FROM AUTHOR]

This chapter provides an overview of the literature in the field of the structural biology of chemokines. The secondary, tertiary, and quaternary structures as determined by x-ray crystallography and nuclear magnetic resonance are compared among the four chemokine families. The biological significance of chemokine structures is explored through a discussion of additional molecules that interact with the chemokines. Specific interactions of chemokines and their receptors are discussed as are interactions between chemokines and glycosaminoglycans. Additionally, a set of tables and figures summarizes the structural information available in the databases. [ABSTRACT FROM AUTHOR]

Chemokines were initially discovered in the context of inflammatory pathologies and were the curiosity of a limited number of researchers. Receptors for these cytokine molecules were identified shortly thereafter and determined to be members of the large G protein-coupled receptor (GPCR) superfamily. Collectively, these basic observations provided a framework to understand mechanisms by which leukocyte subsets could migrate into tissues in a specific manner. Nonetheless, as the field evolved, so came a realization of the broader impact of this family on diverse biological processes, which include the regulation of leukocyte trafficking in hematopoiesis, innate and adaptive immunity, angiogenesis, cancer, and viral pathogenesis. We begin this volume with an overview of research on chemokines and their receptors and then subsequently migrate into a brief discussion of key findings that defined the maturation of this field. As members of the GPCR superfamily have historically dominated drug development programs, so too has interest in chemokine receptors as therapeutic targets. [ABSTRACT FROM AUTHOR]

Although the adrenergic receptors have been cloned since the late 1980s and early 1990s, analysis of the expression of the subtypes in specific tissues and cell types has been hampered because of the lack of high-affinity and high-avidity antibodies. Most of the antibodies currently available have limited use in transfected systems in which receptor density is high but give poor results when used to determine endogenous expression. This limitation in antibody avidity is common in membrane proteins and in G protein-coupled receptors (GPCRs) because of poor protein purification and membrane epitope recognition. Although analysis of ligand binding to tissue homogenates yields an estimate of the tissue content of the various adrenergic receptor subtypes, it does not have sufficient resolution to determine cell type distribution. Currently, methods useful for determining receptor localization are in situ hybridization histochemistry and receptor autoradiography. This chapter reviews the localization of the various adrenergic receptors using these methods. I also introduce the concept and utility of using transgenic mice expressing fluorescenttagged adrenergic receptor subtypes to determine the cell type-specific localization of these receptors using the α1-adrenergic receptor as a model system. [ABSTRACT FROM AUTHOR]

The advent of DNA microarray technology has provided a means to identify changes in the expression of thousands of genes simultaneously. This research tool has enabled investigators to study the effects of adrenergic receptor (AR) stimulation on gene expression on a large scale and has led to the identification of many genes that are regulated by adrenergic receptors (ARs). Microarrays have been used to compare the effects of α1A-AR, α1B-AR, and α1D-AR stimulation on gene expression. This work demonstrated that all three α1-AR subtypes commonly regulate many types of genes. However, genes that are regulated by only one or two α1-AR subtypes have also been identified. These data provide evidence that the physiological roles of the three α1-AR subtypes are not redundant despite their activation by the same ligand, use of common signal transduction pathways, and overlapping tissue distributions. Microarray studies have also identified genes that underlie AR-mediated regulation of the cell cycle, apoptosis, neuronal differentiation, cell hypertrophy, and other biological processes that are regulated by ARs. In addition, microarrays have identified changes in gene expression that accompany AR-mediated disease states, including cardiac hypertrophy, neurodegeneration, and hypermetabolism. The purpose of this chapter is to review how microarrays have contributed to our understanding of AR function at the genomic level. [ABSTRACT FROM AUTHOR]

Natural variations in the genes that encode adrenergic receptors have been identified. The variations of major interest for common diseases are those that occur with allele frequencies of 1% or more and are termed polymorphisms. Of the nine adrenergic receptors (α1A, α1B, α1D, α2A, α2B, α2C, β2C, β1, β2, β3), seven have been found to have nonsynonymous coding polymorphisms. Because of the distribution of adrenergic receptors throughout the body, there is the potential to explore genotype-phenotype associations in many organ systems and diseases. To date, the most extensively studied have been asthma, hypertension, vascular disease, heart failure, and obesity and related metabolic disorders. [ABSTRACT FROM AUTHOR]

Adrenergic receptor (AR) signaling is a key regulator of normal cardiopulmonary homeostasis. Under pathophysiological conditions, such as heart failure, asthma, and hypertension, there are alterations in the signaling cascades. Advances in the ability to manipulate the adenoviral genome have allowed the development of gene therapy in which transgenes of interest are inserted into the adenovirus and transferred to mammals in an organ-specific manner based on delivery methods. These transgenes have included components of the AR signaling pathway that have gone awry at the level of the AR itself or the G protein it activates, the G protein-coupled receptor kinases (GRKs), and regulators of G protein signaling (RGS) proteins that regulate AR desensitization, or the adenylyl cyclase that subsequently activates protein kinase A activity. The use of these vectors in both the heart and the lung has offered promising novel benefits for animal models of disease, including heart failure and lung disorders, and it remains to be determined whether these will be successful future therapeutic strategies in human disease. [ABSTRACT FROM AUTHOR]

Adrenergic receptors (ARs) belong to the largest known family of transmembrane receptors, G protein-coupled receptors (GPCRs). Two subtypes, α and β, have been described, and even though they both respond to norepinephrine and epinephrine, the cellular responses they mediate differ significantly. Many human diseases, such as heart failure, are characterized by alterations in adrenergic signaling. The generation of genetically modified mice with altered expression of one of the AR subtypes has been useful for characterization of the mechanisms of receptor activation, as well as the ensuing in vivo phenotype. The availability of numerous genetically targeted mouse models has been an important tool for the study of AR function and the identification of potential novel therapeutic strategies for a wide range of diseases. [ABSTRACT FROM AUTHOR]

β-Adrenergic receptors (β-ARs) are members of the superfamily of G protein-coupled receptors that are stimulated by the catecholamines epinephrine and norepinephine (1). As part of the sympathetic nervous system, β-ARs have important roles in cardiovascular, respiratory, metabolic, central nervous system, and reproductive functions. Mice lacking one or more of the three β-AR subtype genes (β1, β2, and β3) have been generated to elucidate the physiological role of individual subtypes. Moreover, cells and tissues extracted from these mice have been utilized as tools to understand the molecular and cellular basis of subtype-specific receptor function. These studies are summarized in this chapter. [ABSTRACT FROM AUTHOR]

The α2-adrenergic receptors α2-ARs) belong to the G protein-coupled receptor superfamily and are responsible for mediating a diverse array of physiological effects in multiple tissues in response to the endogenous catecholamines epinephrine and norepinephrine delivered either by synapses or the circulation. Biochemical, physiological, and pharmacological studies have shown that three α2-AR subtypes (α2A, α2B, and α2C) are present in many key target cells and tissues, making them attractive in vivo targets amenable to therapeutic intervention. However, the clarification of the precise in vivo roles attributable to a particular receptor subtype has been complex, largely because of the lack of α2-AR ligands displaying sufficient selectivity among the three α2-AR subtypes. The generation of mice harboring a mutant α2A-AR with diminished capacities (D79N α2A-AR), or null for each of the individual receptor subtype alleles, has yielded a wealth of information critical for the identification and elucidation of their in vivo roles. Further insights have been derived from the creation of mice with combinations of targeted null alleles, mice heterozygous for the α2A-AR, and mice overexpressing the α2C-AR. Collectively, studies in these animals have clarified our understanding of the roles of each receptor subtype; the information revealed en toto exemplifies the power of employing genetically modified mice to elucidate biological functions and reveal effective therapeutic targets. [ABSTRACT FROM AUTHOR]

α1-Adrenergic receptors (ARs) exist in three distinct molecular subtypes: A, B, and D. They are expected to have distinct physiological roles in vivo, but this has been hard to prove because the pharmacological tools to distinguish the subtypes are limited and not always very selective. For this reason, several laboratories have turned to the knockout (KO) approach to dissect the physiological roles of α1-ARs and α1-AR subtypes in vivo. The KOs confirm the importance of α1-ARs in vasoconstriction and blood pressure regulation, but a surprise is the apparently small role of the B in this respect. The D seems limited to a vascular role and probably brain. D-selective antagonists might have an advantage in hypertension treatment if human subtypes are similar to mouse. The B role in glucose metabolism is notable. Perhaps the biggest surprise from the KOs is the essential role of the A or the B in physiological cardiac hypertrophy and cardiac adaptation to stress. The AB KO data provide a plausible mechanism for the adverse effects of nonselective α1-antagonists in clinical trials and raise further concern about use of these drugs in patients with hypertension or prostate disease. [ABSTRACT FROM AUTHOR]

This chapter reviews and compares the strengths, limitations, and potential for development of the two major methodologies employed to visualize the locations and properties of adrenergic receptors: fusion proteins of receptors with fluorescent protein tags and fluorescent ligands. In each case, the three subfamilies of adrenergic receptors (β, α1, and α2) are considered. Emphasis is placed on time sequence imaging in live cells, providing insights to receptor mobilization and regulation. The use of recombinant fusion proteins such as green fluorescent protein has been mainly confined to cell culture; fluorescent ligands can also be utilized in native tissues. It is shown how the two approaches can validate each other and provide complementary information. The importance of appropriate image acquisition and quantitative image analysis is stressed if meaningful data are to be acquired. These approaches have resulted in the recent discovery of more diverse adrenergic receptor locations in both unexpected tissue types and subcellular locations. Finally, the ability to exploit the changing fluorescent properties of interacting fluorophores to elucidate interactions between receptor molecules, such as dimerization or chaperoning, are considered. It is concluded that we have entered a new phase in which visualization of adrenergic receptors in multidimensional analysis is providing new insights to receptor biology, and that visualization in live heterogeneous organs and tissues is bringing this to the physiological level. [ABSTRACT FROM AUTHOR]

The sympathetic nervous system is a main regulator of homeostasis and mediates most of its effects via the nine subtypes of α1-, α2-, and β-adrenergic receptors. Although recent years have witnessed major progress in the identification of adrenergic receptor subtypes with specific cell and tissue functions in experimental animals, similar progress for human tissues remains limited. At present, most data are available for β-adrenergic receptor subtypes in the human heart, but substantial information is available regarding urogenital tissues, particularly the prostate and the bladder. The discovery of a specific role of α1A-adrenergic receptors in the human prostate has led to the development of selective antagonists that effectively treat prostate symptoms without major effects on blood pressure. Hence, α1-adrenergic antagonists have largely replaced surgery as the primary treatment of the highly prevalent condition of benign prostatic hyperplasia. It is hoped that better phenotyping of other human tissues will contribute to similar progress in other disease states. [ABSTRACT FROM AUTHOR]

The family of adrenergic receptors (ARs; the α1-, α2-, and β-ARs) are key regulators of the sympathetic division of the autonomic nervous system, involved in both central and peripheral cardiovascular function. Here, we review our current understanding of the cellular localization and trafficking properties of the α1-, α2-, and β-ARs. We then examine recent evidence indicating that the cellular localization of these receptors and their excursion into intracellular compartments play an underappreciated role in the activation of both G protein and novel non-G protein-dependent cellular signaling. [ABSTRACT FROM AUTHOR]

Adrenergic receptors are critical mediators of sympaiMolecular Cardiology Program, Vihysiological responses. Activated by the neurotransmitter and neurohormone, norepinephrine and epinephrine, released from sympathetic nerve endings and the adrenal medulla, respectively, they play a central role in this evolutionarily ancient defense system that regulates many physiological functions, including those involved in circulatory, metabolic, respiratory, and central nervous system homeostasis. In addition, alterations in the regulation and molecular structure of adrenergic receptors have been implicated in a variety of diseases, and drugs targeting these receptors are important and widely used therapeutics. The molecular cloning of the first adrenergic receptor in 1986 revealed structural homology with the functionally related rhodopsin visual transduction system—a finding that led to the realization that these receptors formed a new superfamily of proteins, now known as G protein-coupled receptors. Since that time, a plethora of structure-function studies have provided major insights into the molecular determinants of adrenergic receptor ligand-binding, activation, and signaling, many of which are relevant not only to the adrenergic receptor family but also, more generally, to the broader superfamily of G protein-coupled receptors. These advances in our understanding of adrenergic receptor activation, regulation, and functioning are reviewed in this chapter. [ABSTRACT FROM AUTHOR]

All adrenergic receptors (ARs) are members of the G protein-coupled receptor superfamily and have been assumed to initiate signals primarily by activation of heterotrimeric G proteins. The three major AR families (α1, α2, β) each contain three subtypes, with all receptors within a subfamily acting through the same G proteins to initiate the same signals. α1-ARs activate Gq/11 to increase Ca2+, α2-ARs activate Gi/o to decrease cyclic adenosine 5′-monophosphate, and β-ARs activate Gs to increase cyclic adenosine 5′-monophosphate. This raises questions regarding how apparently redundant receptor subtypes have survived evolutionarily and continue to mediate distinct functions in all known higher organisms. Although the primary importance of G proteins in signaling is not in doubt, it is increasingly clear that understanding AR signaling requires additional complexity. ARs have now been shown also to interact directly with other proteins, which may be important in signaling. One class includes other G protein-coupled receptors, and increasing reports of receptor heterodimerization are transforming our view of these receptors as solitary cellular sentinels for detecting incoming signals. Another class is adaptor or scaffolding proteins responsible for local organization of specific signaling complexes, for which proximity of effector molecules may result in increased or unexpected responses. Other proteins, such as regulators of G protein signaling, may affect the specificity or extent of G protein activation. Finally, internalization of receptors may be required for certain responses, which may be independent of G protein signaling. Thus, the traditional view of a linear signaling cascade of ligand/receptor/G protein/second messenger activation is turning into a much more complex, combinatorial, and context-dependent view of AR signaling. [ABSTRACT FROM AUTHOR]

During the 20th century, extraordinary progress was made in our understanding of adrenergic receptors. This progress was the result of the hard work and insightful thinking of a remarkable cadre of investigators throughout the world. A summary of some of the more important developments is presented two ways: as a summary listing by decade and as four major, overlapping eras—biochemical, physiological, pharmacological, and molecular. [ABSTRACT FROM AUTHOR]