1994 — 1998 |
Von Zastrow, Mark E. |
K21Activity Code Description: To foster the development of outstanding scientists with potential for making important contributions to the fields of alcoholism, drug abuse or mental health (ADM) research. Primarily intended to meet the need for supervised research experience for highly promising biological or behavioral scientists who need further supervised research experience. |
Mechanisms of Receptor Internalization in Drug Addiction @ University of California San Francisco
This is a request for a scientist Development Award to support training in molecular biological and biochemical approaches for studying receptor.based mechanisms of drug addiction The prolonged administration of certain addictive drugs causes sequestration and/or down regulation of G protein-coupled receptors. These phenomena involve drug-induced internalization of receptors from the plasma membrane and their delivery to specific populations of intracellular vesicles. The mechanisms that mediate and control this receptor internalization are not known. While several classes of cellular proteins have been identified that associate with receptors and mediate specific events in signal transduction, studies of mutant receptors and cell lines indicate that none of these associations is required for drug-induced receptor internalization. Therefore other, as yet unidentified, mechanisms must mediate and control drug-regulated internalization. By focusing on adrenergic receptors as a physiologically important and experimentally advantageous model system, the proposed studies will provide training in biochemical and molecular biological approaches to (1) define receptor domains that determine specific steps in the internalization mechanism, (2) identify cellular proteins that associate with receptors and mediate these steps, and (3) purify and clone these additional proteins. This work will elucidate, in molecular detail, fundamental biological mechanisms relevant to drug addiction.
|
0.958 |
1997 — 2008 |
Von Zastrow, Mark E. |
R29Activity Code Description: Undocumented code - click on the grant title for more information. R37Activity Code Description: To provide long-term grant support to investigators whose research competence and productivity are distinctly superior and who are highly likely to continue to perform in an outstanding manner. Investigators may not apply for a MERIT award. Program staff and/or members of the cognizant National Advisory Council/Board will identify candidates for the MERIT award during the course of review of competing research grant applications prepared and submitted in accordance with regular PHS requirements. |
Membrane Trafficking of Opioid Receptors @ University of California San Francisco
DESCRIPTION (provided by applicant): A fundamental mechanism by which opioid receptors are regulated is by rapid endocytosis after agonistinduced activation. Receptors traverse divergent intracellular membrane pathways after their initial endocytosis, and receptor "sorting" between distinct downstream pathways plays a critical role in determining the functional consequences of endocytosis. The proposed studies seek to elucidate mechanisms that determine whether endocytosed opioid receptors recycle rapidly to the cell surface (a well characterized pathway promoting functional recovery or "resensitization" of signal transduction), or if they traffic to lysosomes (a well established pathway of proteolytic "down-regulation" that leads to a prolonged attenuation of signal transduction). The Specific Aims of the proposed studies are to: (1) Define the biochemical properties of a mechanism that mediates signal-dependent recycling of opioid receptors; (2) Elucidate a distinct mechanism that promotes sorting of endocytosed opioid receptors to lysosomes; (3) Determine whether opioid receptors are sorted between distinct membrane domains of multivesicular endosomes; and (4) Investigate the functional relevance of specific post-endocytic sorting mechanisms to opioid receptor regulation in neurons. These studies have general relevance to understanding mechanisms by which G protein-coupled receptors are regulated and specific relevance to mechanisms underlying physiological adaptation of the nervous system in response to clinically important opiate drugs.
|
0.958 |
1999 — 2003 |
Von Zastrow, Mark E. |
K02Activity Code Description: Undocumented code - click on the grant title for more information. |
Novel Proteins That Regulate Opioid Receptors @ University of California San Francisco
Opioid receptors undergo rapid, ligand-dependent endocytosis by clathrin-coated pits. Mounting evidence implicates this process as a fundamental component of the complex physiological regulation of opioid receptors, which is induced by native opioid peptides and distinguishes the actions of addictive opiate drugs. Therefore, understanding molecular mechanisms that control this process has broad relevance to opioid biology and addiction research. One mechanism by which ligands control the entry of opioid receptors into clathrin-coated pits is mediated by the phosphorylation-dependent association of agonist-activated receptors with beta-arrestin. Preliminary studies suggest the existence of an additional mechanism of endocytic regulation, which is expressed in a cell type-specific manner and appears to be mediated by novel protein interactions. The studies proposed in this application are designed to identify specific proteins that mediate this regulatory mechanism and examine the function of these proteins in several cell types. The Specific Aims of the proposed studies are to (1) define cytoplasmic residues required for cell type-specific endocytosis of a truncated mutant opioid receptor, (2) identify and clone cellular proteins which interact with the defined receptor domain(s), and (3) elucidate the functional role of selected interacting proteins in regulating opioid receptor endocytosis. These studies will contribute to a fundamental molecular understanding of mechanisms that regulate opioid receptors and mediate cell type-specific differences in receptor regulation. Thus the proposed research is directly relevant to understanding the cellular physiology of opiate drug action and addiction in complex neural tissue.
|
0.958 |
2000 — 2004 |
Von Zastrow, Mark E. |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Proteins Regulating Endocytosis of Opioid Receptors @ University of California San Francisco
Endocytosis of opioid receptors modulates signal transduction and is thought to be involved in opiate drug action and addiction. This process is highly regulated by ligands, both in cultured cells and native tissues. A phosphorylation-dependent mechanism that promotes receptor endocytosis has been described previously. Preliminary studies suggest the hypothesis that additional mechanisms contribute to the physiological regulation of opioid receptor endocytosis observed in intact cells. Furthermore, the preliminary data suggest that these mechanisms are relevant to opiod receptor regulation in neurons. The proposed studies test this hypothesis by focusing on two distinct mechanisms, which have opposite effects on receptor endocytosis and differ in their dependence on receptor phosphorylation. The proposed experiments seek to elucidate these mechanisms in biochemical detail, identify cellular proteins that mediate them, and examine their functional consequences in neurons. The Specific Aims are to (1) characterize a mechanism that promotes rapid endocytosis of a truncated, phosphorylation-deficient mutant opioid receptor; (2) characterize a phosphorylation-regulated mechanism that inhibits endocytosis of full-length opioid receptors; and (3) identify cellular proteins that interact specifically with defined receptor domains and elucidate their role in regulating endocytosis of opioid receptors in several cell types. These studies have direct relevance to mechanisms underlying opiate drug action and addiction. In addition, they may be relevant to understanding alternate mechanisms regulating endocytosis of other G protein-coupled receptors.
|
0.958 |
2004 |
Von Zastrow, Mark E. |
P41Activity Code Description: Undocumented code - click on the grant title for more information. |
Phosphorylative Decoding of Opiate Interactions Using Ms @ University of California San Francisco |
0.958 |
2005 — 2008 |
Von Zastrow, Mark E. |
P41Activity Code Description: Undocumented code - click on the grant title for more information. |
Phosphorylative Decoding of Opiate Interactions Using Mass Spectrometry @ University of California San Francisco |
0.958 |
2005 — 2008 |
Von Zastrow, Mark E. |
K02Activity Code Description: Undocumented code - click on the grant title for more information. |
Post-Endocytic Sorting of Opioid Receptors @ University of California San Francisco
DESCRIPTION (provided by applicant): This is an application for a renewal of a NIDA sponsored K02 Independent Scientist Award. G protein-coupled receptors (GPCRs) comprise the largest known family of signaling receptors, mediate diverse functions in the CNS, are critical targets for drugs of abuse, and are extensively regulated in vivo. One mechanism of receptor regulation, on which clinically relevant drugs can have diverse effects, involves regulated membrane trafficking of receptors through the endocytic pathway. In previous studies a specific mechanism by which opioid neuropeptide receptors undergo initial endocytosis from the plasma membrane was identified and investigated. The current proposal seeks to elucidate mechanisms by which the endocytic membrane trafficking of G protein-coupled opioid receptors is controlled after endocytosis. The Specific Aims of the proposed studies are to: (1) Define the biochemical properties of a mechanism that mediates signal-dependent recycling of opioid receptors; (2) Elucidate a distinct mechanism that promotes sorting of endocvtosed opioid receptors to lysosomes; (3) Determine whether opioid receptors are sorted between distinct membrane domains of multivesicular endosomes; and (4) Investigate the functional relevance of specific post-endocytic sorting mechanisms to opioid receptor regulation in neurons. These studies have general relevance to understanding mechanisms by which G protein-coupled receptors are regulated, and will contribute fundamental information to understanding physiological adaptation of the nervous system in response to clinically important opiate drugs.
|
0.958 |
2006 — 2008 |
Von Zastrow, Mark E. |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Endocytosis of Mesolimbic Opioid and Dopamine Receptors @ University of California San Francisco |
0.958 |
2006 — 2008 |
Von Zastrow, Mark E. |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Mechanisms Regulating Endocytosis of Opioid Receptors @ University of California San Francisco
[unreadable] DESCRIPTION (provided by applicant): Opioid receptors are members of the large superfamily of G protein-coupled receptors (GPCRs), and are activated both by endogenously produced opioid neuropeptides and by a diverse group of addictive non- peptide drugs. Endogenous opioid peptides induce a complex series of regulatory processes, which begin with rapid phosphorylation and endocytosis of ligand-activated receptors. Non-peptide opioid drugs, including certain important opiate alkaloid such as morphine, have been observed to induce these regulatory processes to a smaller degree than opioid peptide agonists. Little is known about specific phosphorylation reactions occurring on the wild type receptor in intact cells. Furthermore, the functional effects of specific phosphorylation reactions on receptor regulation in physiologically relevant CNS neurons are not defined. The proposed studies address this question using novel protein biochemical, mass spectrometric and chemical genetic approaches. The Specific Aims are to 1) Define the effects of selected agonist ligands on regulatory phosphorylation of wild type receptors in intact cells; 2) Define the functional significance of distinct phosphorylated receptor species to agonist-selective regulation of receptors; 3) Elucidate a novel mechanism by which transient elevation of GRK2 activity produces a sustained increase in morphine- induced uOR endocytosis; and 4) Determine the importance of receptor regulatory mechanisms defined in HEK293 cells to morphine-induced endocytosis in cultured medium spiny neurons. These studies are specifically relevant to the fields of opioid biology and addiction research. They may also have more general implications for understanding ligand-dependent regulation of other GPCRs that, as a family, comprise the largest single group of clinically useful drug targets. [unreadable] [unreadable] [unreadable]
|
0.958 |
2007 — 2008 |
Von Zastrow, Mark E. |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Endocytosis Mesolimbic Opioid and Dopamine Receptors @ University of California San Francisco |
0.958 |
2007 |
Von Zastrow, Mark E. |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Extracellular Matrix Protein Enhancement of Phagocytosis @ University of California San Francisco
[unreadable] DESCRIPTION (provided by applicant): It is now well accepted that an important aspect of leukocyte interaction with extracellular matrix is that matrix proteins induce cell activation. The rationale for this project is to understand the molecular mechanisms involved in this mechanism of leukocyte activation. Several years ago we found that in addition to integrins, a non-integrin protein of the phagocyte plasma membrane was required for extracellular matrix induced activation. We called this IgV superfamily member with multiple transmembrane segments Integrin-associated Protein (lAP) because of its physical and functional association with phagocyte integrins. Subsequently, we showed that this protein is identical to CD47. Now it is clear from our work and others that CD47 has roles in several important processes in inflammation and immunity, including activation of phagocytosis, respiratory burst, and integrin-mediated adhesion; leukocyte transendothelial and transepithelial migration; synergy with TCR in lymphocyte activation; and even regulation of lymphocyte apoptosis. Recently, we have focused on understanding the molecular mechanisms by which CD47 affects this panoply of functions in diverse cell types. We have demonstrated CD47 association with heterotrimeric G proteins and shown that both these G proteins and rho family G proteins regulate CD47 function. We also have shown that CD47 associates with p21-activated kinase (pak) and with a novel cytoplasmic protein that regulates heterotrimeric G protein signaling, PLIC-1. These data suggest the hypothesis that a fundamental role for CD47 is to act as both an effector and a regulator of G protein signaling. The next funding period will be devoted to testing this hypothesis and its implications for how CD47 regulates leukocyte activation. [unreadable] Specifically, I propose to determine :1. What are the structural requirements in CD47 for GTPase [unreadable] regulation?; 2. What are the roles for rho family GTPases and pak in CD47 functions?; and 3) How does the CD47-associated cytosolic protein PLIC-1 regulate Gi signaling? These studies will contribute greatly to the understanding and ultimately the control of inflammatory and immune responses. [unreadable] [unreadable] [unreadable] [unreadable]
|
0.958 |
2007 — 2008 |
Von Zastrow, Mark E. |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Gpcr Regulation in Nociceptors @ University of California San Francisco
Opioid analgesic drugs modulate primary nociceptive neurons by activating mu (MOR) and delta (DOR) - type G protein-coupled opioid receptors. The beta-2 adrenergic receptor (B2AR) is a distinct G protein- coupled receptor expressed in nociceptive neurons that can produce enhanced, rather than reduced, nociceptive responses. Studies of heterologous cell models indicate that both opioid and adrenergic receptors are regulated by a shared mechanism, involving receptor phosphorylation by G protein-coupled receptor kinases (GRKs) followed by rapid endocytosis. Little is known about the occurrence or function of this conserved regulatory mechanism in nociceptive neurons. A combination of immunohistochemical and live cell imaging approaches will be used to investigate the steady state distribution and regulated endocytic trafficking of MOR, DOR and B2AR in nociceptive neurons, both in the intact tissue and in dissociated neuronal culture. The effects of selected opioid and adrenergic agonists on regulated endocytosis of receptors will be determined using quantitative trafficking assays. The possible functional importance of a specific mechanism of receptor sorting after endocytosis, which determines the signaling consequences of regulated endocytosis in heterologous cell models, will be probed in nociceptive neurons using previously established mutations. A novel protein engineering method will be applied to temporally control GRK activity in nociceptive neurons, and to develop a new approach potentially suitable for probing functional effects on nociceptive signaling in vivo. These studies will provide fundamental insight to cell biological mechanisms relevant to opioid analgesia and tolerance. They may also provide insight to mechanisms that modulate hyperalgesia produced by adrenergic activation in nociceptive neurons.
|
0.958 |