Showing papers by "Robert J. Lefkowitz published in 2011"

Distinct phosphorylation sites on the β(2)-adrenergic receptor establish a barcode that encodes differential functions of β-arrestin.

Abstract: Phosphorylation of G protein–coupled receptors (GPCRs, which are also known as seven-transmembrane spanning receptors) by GPCR kinases (GRKs) plays essential roles in the regulation of receptor function by promoting interactions of the receptors with β-arrestins. These multifunctional adaptor proteins desensitize GPCRs, by reducing receptor coupling to G proteins and facilitating receptor internalization, and mediate GPCR signaling through β-arrestin–specific pathways. Detailed mapping of the phosphorylation sites on GPCRs targeted by individual GRKs and an understanding of how these sites regulate the specific functional consequences of β-arrestin engagement may aid in the discovery of therapeutic agents targeting individual β-arrestin functions. The β 2 -adrenergic receptor (β 2 AR) has many serine and threonine residues in the carboxyl-terminal tail and the intracellular loops, which are potential sites of phosphorylation. We monitored the phosphorylation of the β 2 AR at specific sites upon stimulation with an agonist that promotes signaling by both G protein–mediated and β-arrestin–mediated pathways or with a biased ligand that promotes signaling only through β-arrestin–mediated events in the presence of the full complement of GRKs or when either GRK2 or GRK6 was depleted. We correlated the specific and distinct patterns of receptor phosphorylation by individual GRKs with the functions of β-arrestins and propose that the distinct phosphorylation patterns established by different GRKs establish a “barcode” that imparts distinct conformations to the recruited β-arrestin, thus regulating its functional activities.

Quantifying ligand bias at seven-transmembrane receptors.

Abstract: Seven transmembrane receptors (7TMRs), commonly referred to as G protein-coupled receptors, form a large part of the “druggable” genome. 7TMRs can signal through parallel pathways simultaneously, such as through heterotrimeric G proteins from different families, or, as more recently appreciated, through the multifunctional adapters, β-arrestins. Biased agonists, which signal with different efficacies to a receptor9s multiple downstream pathways, are useful tools for deconvoluting this signaling complexity. These compounds may also be of therapeutic use because they have distinct functional and therapeutic profiles from “balanced agonists.” Although some methods have been proposed to identify biased ligands, no comparison of these methods applied to the same set of data has been performed. Therefore, at this time, there are no generally accepted methods to quantify the relative bias of different ligands, making studies of biased signaling difficult. Here, we use complementary computational approaches for the quantification of ligand bias and demonstrate their application to two well known drug targets, the β2 adrenergic and angiotensin II type 1A receptors. The strategy outlined here allows a quantification of ligand bias and the identification of weakly biased compounds. This general method should aid in deciphering complex signaling pathways and may be useful for the development of novel biased therapeutic ligands as drugs.

A stress response pathway regulates DNA damage through β2-adrenoreceptors and β-arrestin-1.

Abstract: The human mind and body respond to stress, a state of perceived threat to homeostasis, by activating the sympathetic nervous system and secreting the catecholamines adrenaline and noradrenaline in the 'fight-or-flight' response. The stress response is generally transient because its accompanying effects (for example, immunosuppression, growth inhibition and enhanced catabolism) can be harmful in the long term. When chronic, the stress response can be associated with disease symptoms such as peptic ulcers or cardiovascular disorders, and epidemiological studies strongly indicate that chronic stress leads to DNA damage. This stress-induced DNA damage may promote ageing, tumorigenesis, neuropsychiatric conditions and miscarriages. However, the mechanisms by which these DNA-damage events occur in response to stress are unknown. The stress hormone adrenaline stimulates β(2)-adrenoreceptors that are expressed throughout the body, including in germline cells and zygotic embryos. Activated β(2)-adrenoreceptors promote Gs-protein-dependent activation of protein kinase A (PKA), followed by the recruitment of β-arrestins, which desensitize G-protein signalling and function as signal transducers in their own right. Here we elucidate a molecular mechanism by which β-adrenergic catecholamines, acting through both Gs-PKA and β-arrestin-mediated signalling pathways, trigger DNA damage and suppress p53 levels respectively, thus synergistically leading to the accumulation of DNA damage. In mice and in human cell lines, β-arrestin-1 (ARRB1), activated via β(2)-adrenoreceptors, facilitates AKT-mediated activation of MDM2 and also promotes MDM2 binding to, and degradation of, p53, by acting as a molecular scaffold. Catecholamine-induced DNA damage is abrogated in Arrb1-knockout (Arrb1(-/-)) mice, which show preserved p53 levels in both the thymus, an organ that responds prominently to acute or chronic stress, and in the testes, in which paternal stress may affect the offspring's genome. Our results highlight the emerging role of ARRB1 as an E3-ligase adaptor in the nucleus, and reveal how DNA damage may accumulate in response to chronic stress.

Multiple ligand-specific conformations of the β2-adrenergic receptor

Abstract: Seven-transmembrane receptors (7TMRs), also called G protein-coupled receptors (GPCRs), represent the largest class of drug targets, and they can signal through several distinct mechanisms including those mediated by G proteins and the multifunctional adaptor proteins β-arrestins. Moreover, several receptor ligands with differential efficacies toward these distinct signaling pathways have been identified. However, the structural basis and mechanism underlying this 'biased agonism' remains largely unknown. Here, we develop a quantitative mass spectrometry strategy that measures specific reactivities of individual side chains to investigate dynamic conformational changes in the β(2)-adrenergic receptor occupied by nine functionally distinct ligands. Unexpectedly, only a minority of residues showed reactivity patterns consistent with classical agonism, whereas the majority showed distinct patterns of reactivity even between functionally similar ligands. These findings demonstrate, contrary to two-state models for receptor activity, that there is significant variability in receptor conformations induced by different ligands, which has significant implications for the design of new therapeutic agents.

β-Arrestin Deficiency Protects Against Pulmonary Fibrosis in Mice and Prevents Fibroblast Invasion of Extracellular Matrix

Abstract: Idiopathic pulmonary fibrosis is a progressive disease that causes unremitting extracellular matrix deposition with resulting distortion of pulmonary architecture and impaired gas exchange. β-Arrestins regulate G protein (heterotrimeric guanine nucleotide-binding protein)-coupled receptors through receptor desensitization while also acting as signaling scaffolds to facilitate numerous effector pathways. Here, we examine the role of β-arrestin1 and β-arrestin2 in the pathobiology of pulmonary fibrosis. In the bleomycin-induced mouse lung fibrosis model, loss of either β-arrestin1 or β-arrestin2 resulted in protection from mortality, inhibition of matrix deposition, and protected lung function. Fibrosis was prevented despite preserved recruitment of inflammatory cells and fibroblast chemotaxis. However, isolated lung fibroblasts from bleomycin-treated β-arrestin-null mice failed to invade extracellular matrix and displayed altered expression of genes involved in matrix production and degradation. Furthermore, knockdown of β-arrestin2 in fibroblasts from patients with idiopathic pulmonary fibrosis attenuated the invasive phenotype. These data implicate β-arrestins as mediators of fibroblast invasion and the development of pulmonary fibrosis, and as a potential target for therapeutic intervention in patients with idiopathic pulmonary fibrosis.

Dopaminergic Receptors in the Anterior Pituitary Gland

Abstract: MARC G. CARON,+ MICH~LE BEAULIEU,~ VINCENT RAYMOND,~ BERNARD GAGN~, JACQUES DROUIN,§ ROBERT J. LEFKOWITZ,~ AND FERNAND LABRIE/~ From the Medical Research Council Group in Molecular Endocrinology, Le Centre Hospitalier de 1’Universite Laval, Quebec GlV 4G2 and Departments of Medicine and Biochemistry, Duke University Medical Center, Durham, North Carolina 27710 The ergot alkaloid [“Hldihydroergocryptine, a potent do- paminergic agonist, has been used to study binding sites in bovine anterior pituitary membranes. One function of the anterior pituitary gland, prolactin secretion, as we show is under typical dopaminergic control and was measured in uitro in another series of experiments using rat anterior pituitary cells

Introduction to the Series on Novel Aspects of Cardiovascular G-Protein-Coupled Receptor Signaling

Abstract: The initial concept of drugs acting on receptors is generally credited to John Newport Langley (1852–1925) and his pioneering work on the antagonistic effects of atropine,1 and to Paul Ehrlich (1854–1915), who coined the word “receptor.”2 It was in 1905 that Langley (together with the much forgotten Thomas Renton Elliott) proposed that a “receptive substance” was the site of action of chemical mediators liberated by nerve stimulation.3 At approximately the same time, Ehrlich in his studies on immunity postulated that a drug could have a therapeutic effect only if it “unites with certain chemical groupings in the protoplasm of cells” and called such cell groupings “poison receptors” or just “receptors.”2 In experiments performed in the early 1900s, the English pharmacologist and neurophysiologist Sir Henry Hallett Dale (1875–1968) identified the muscarinic and cholinergic actions of acetylcholine and subsequently shared with Otto Loewi the Nobel Prize in 1936 for discoveries relating to the role of acetylcholine in the chemical transmission of nerve impulses.4 Dale coined the terms “adrenergic” and “cholinergic” to describe the actions of autonomic and motor nerve fibers, and he recognized that the sympathetic myoneural junction could also be called “the receptive mechanism for adrenaline.”5 Based on Dale's experiments on the physiological effect of Ergot preparations, the concept evolved that there were two classes of adrenotropic receptors: those whose action results in excitation and those whose action results in inhibition of the effector cells.5,3 This concept of two classes of adrenotropic receptors was further clarified by Raymond Ahlquist at the Medical College of Georgia, when in 1948 he published his work showing that it is an oversimplification to classify adrenotropic receptors as either excitatory or inhibitory.6 By studying the relative potencies of six sympathomimetic amines on several isolated mammalian preparations, …

A tale of two callings

Abstract: First, I want to thank Ralph for that wonderful presentation. After listening to all that, I can hardly wait to hear what I am going to say. Preparing these Kober Medal presentations requires a tremendous amount of work. When I first asked Ralph if he would do me the honor of making these remarks, he asked if I had any guidelines I wanted to give him. I responded: Ralph you know that I would want something simple and modest — not overly ostentatious or hagiographic — no matter how long it takes. Seriously though, Ralph has been my closest friend for more than 30 years, and I am in his debt for this. All the more meaningful to share this wonderful moment with a number of family members, including, amongst others, three of my five children, two of my four grandchildren, some in-laws, and my wife, Lynn. As they say, behind every successful man stands a surprised woman. Her constant and loving support is one of my true blessings. The Kober Medal has such a special place in our profession that, despite more than a year to accomplish the task, I continue to have difficulty getting my head around the notion that I am joining the ranks of its recipients. For the more than 40 years that I have attended these meetings, the Kober Medal presentation to a series of individuals I viewed as iconic heroes has always been a highlight for me, something I would never miss, a yearly source of inspiration and delight. So, despite my very real qualms about assuming a place in this pantheon, I will for the moment adopt the philosophy of “fake it until you make it.” As a young child growing up in New York City, I was very prone to hero worship. I mention this because I have been struck in recent years by a remarkable commonality apparent in the earliest aspirations of several Kober medalists as presented at this meeting. This picture depicts a young Joe Goldstein, a recent Kober medalist and former Nobel laureate, that was shown by Jean Wilson when he presented the Kober Medal to Mike Brown and Joe a few years back (Figure ​(Figure1).1). Unlike myself, Joe grew up in a small town in South Carolina. Now, compare this photo of me taken at about the same time on the streets of the Bronx. This series of N = 2 has suggested to me that an early admiration for Western heroes may be a stepping stone to the Kober Medal later in life, regardless of the widely divergent cultural context in which the winners may have grown up. Figure 1 Two future Kober Medalists: Joe “Butch” Goldstein (left) and Bob “Sundance” Lefkowitz (right). At about the same time that these photos were taken, one of my most important heroes was my family physician, a man named Dr. Joseph Feibush. He was a general practitioner who made house calls and made me feel better when I was sick. By the third grade I was quite convinced I wanted to grow up to be just like him, a practicing physician who healed the sick. From then on I also loved reading books about doctors, especially novels in which an MD played a central, usually heroic, role. This brings me to another point — how remarkably fortunate I have been to experience my life’s work as a calling. Not only did this sense of a calling to clinical medicine clarify and direct my early years through medical school and residency, but it certainly saved me all the anguish that many young people seem to face in finding their way in adult society. But I have been doubly fortunate in this regard in that I would subsequently feel a second calling, one to scientific research. I would never have imagined this during medical school and house staff days. I was totally devoted to clinical work and avoided all research electives in medical school to focus on clinical activities. Nonetheless, two of my professors at Columbia College of Physicians and Surgeons who had the most pronounced influence on me were Paul Marks and Dickinson Richards, himself a former Kober and Nobel medalist for his role in developing cardiac catheterization. These two men introduced me to a way of bringing scholarly scientific findings to the bedside of sick patients, which really began to awaken a dormant scientific curiosity in me. As with so many physicians of my generation, my two-year experience as an officer in the United States Public Health Service at the NIH from 1968 to 1970 in fulfillment of my draft obligation forever altered the path of my career. In the laboratory, I made a remarkably slow start. Lacking in basic laboratory experience and technique as well as the necessary patience and perspective, my fledgling attempts at research met with unrelenting failure during my first 18 months there. It was during this time of failure and frustration that I made arrangements to return to full-time residency and then cardiology fellowship at the MGH to follow my two-year stint in Bethesda. I had never really failed at anything before so this was a new experience for me, but one which would help me in later years to advise my own trainees. However, I was fortunate during this period to have two wonderful mentors, both members of this association. My spirits were continuously elevated by the unflagging and buoyant enthusiasm of Jesse Roth, even while I was continuously brought down to earth by the rigorous temperament of Ira Pastan, who never failed to point out the key controls missing from my experimental design, which generally invalidated my conclusions. Somewhere around the 18-month mark, I finally began to make some progress, and things started to turn around. Though tempted to extend my time at the NIH, I honored my commitment to the MGH, and so after the two-year assignment, I headed off to residency again. The next six months were absolutely pivotal for me. I threw myself into the clinical work with my usual fervor. But something was missing. For the first time in several years I had no data. I was like a junkie who needed a fix. As I thought back to the dark days of my failing experiments during my first year at the NIH, I had the epiphany that even negative data was better than no data. The second six months of my senior residency year was supposed to consist of elective rotations. House rules prohibited residents from working in research labs since they were paid with clinical dollars. Nonetheless I arranged to surreptitiously do research in the lab of the late Edgar Haber, Chief of Cardiology. His labs were located deep in the basement of the Bullfinch Building. I got away with it for a while. Then, late one cold winter night, the residency director, Dan Federman, was taking a shortcut to the parking lot through tunnels that wound past Ed’s lab. He caught me walking in the hallway carrying a rack of test tubes. Waving a finger in my face, he said, “Lefkowitz, I heard rumors that you were doing research, see me in my office tomorrow.” The next day, he and Alex Leaf, Chairman of the Department of Medicine, gently upbraided me, but it was just a slap on the wrist, and they never really ordered me to desist, which is what I had feared. I continued my research in Ed’s lab throughout my cardiology fellowship over the next two years, and it was here that I initiated my work on the adrenergic receptors. I moved to Duke in June of 1973, recruited by the Chief of Cardiology, Andy Wallace, and the Chairman of Medicine, Jim Wyngaarden. They wanted me to start a program in Molecular Cardiology. They both kept a helpful and supportive eye on me the first few years, reading my grant applications and in Jim’s case actually reviewing my research findings a couple of times a year. I would be remiss if I did not acknowledge the two major sources which have funded my research. First is the NIH R01 grant mechanism. My first R01 has been active for almost 40 years now. And then, of course, there is the Howard Hughes Medical Institute (HHMI). I became a Hughes investigator in 1976, so that my tenure with the organization is now 35 years and counting. I have served under every research director and president that the institute has had, beginning with George Thorn. It is truly a remarkable organization which has enabled much of what I have been able to accomplish. Alas, however, fewer and fewer of the investigators today are physicians. It’s hard for me to believe that I have been at Duke for almost 40 years now. During that time about 200 fellows and students have worked with me in the laboratory. A dozen of these are members of this association. Mentoring these trainees has been one of the greatest joys of my professional career. And I watch their independent careers evolve with extraordinary pride in their subsequent accomplishments. And, on this note, it seems to me appropriate that I conclude these brief remarks by highlighting this extraordinary group of individuals. This photo was taken eight years ago when about half of my trainees up to that time returned for my 60th birthday party (Figure ​(Figure2).2). As you look at this, I think you will be able to understand what is undoubtedly at the core of whatever success I may have achieved in my career. It is simply this: “Nothing is impossible for the man who doesn’t have to do it for himself.” Thank you very much. Figure 2 “Nothing is impossible for the man who doesn’t have to do it for himself.”

COMPOSITIONS FOR BINDING β-ARRESTIN, AND THEIR USE TO MODULATE β-ARRESTIN ACTIVITY

Abstract: The present invention relates in general to modulation of β-arrestin2 activity, and, in particular, methods and compositions to modulate β-arrestin2 activity in leukemic cells in the CML disease process to inhibit CML disease.