Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-04-20
2003-02-04
Eyler, Yvonne (Department: 1696)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S069100, C435S091200, C435S173300, C435S252300, C435S320100, C536S023100, C536S024300, C530S300000, C530S350000
Reexamination Certificate
active
06514696
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to a G protein-coupled receptor which is expressed predominantly in lymphoid cells, is transcriptionally induced in response to proliferative stimuli and genotoxic treatment and integrates diverse cellular signals by modulation of cytoskeletal architecture.
BACKGROUND OF THE INVENTION
The family of G protein-coupled receptors (GPCRs) has at least 250 members (Strader et al.
FASEB J
., 9:745-754, 1995; Strader et al.
Annu. Rev. Biochem
., 63:101-32, 1994). It has been estimated that one percent of human genes may encode GPCRs. GPCRs bind to a wide-variety of ligands ranging from photons, small biogenic amines (i.e., epinephrine and histamine), peptides (i.e., IL-8), to large glycoprotein hormones (i.e., parathyroid hormone). GPCRs play important roles in diverse cellular processes including cell proliferation and differentiation, leukocyte migration in response to inflammation, and cellular response to light, odorants, neurotransmitters and hormones (Strader et al., supra.).
Ligand binding to GPCRs elicits activation of signaling pathways via associated heterotrimeric G proteins comprising &agr;, &bgr; and &ggr; subunits. Heterotrimeric G proteins are classified according to the &agr; subunit, which can be any of more than 20 grouped into 4 classes termed G
s
, G
i
, G
q
and G
12
. Similarly, there are 6 known &bgr; and 12 &ggr; subunits, further increasing the complexity, although not all possible combinations are functional (Schmidt et al.,
J. Biol. Chem
. 267:13807-13810, 1992). Initiation of signal transduction follows exchange of GTP for GDP bound to the &agr;-subunit and its dissociation from the &bgr;&ggr; dimer. Both released GTP-bound &agr; and free &bgr;&ggr; components are capable of mediating signaling events through their interaction with effector molecules, leading to an appropriate physiological response. Importantly, activation of G protein signaling is integrated with receptor downregulation via serine/threonine phosphorylation of critical residues within the carboxy terminal cytoplasmic tail of the GPCR by GPCR kinases (GRKs) as well as Protein Kinase A (PKA) and Protein Kinase C (PKC) leading to desensitization and internalization of the receptor (Ferguson et al.,
Biochem. Soc. Trans
. 24:953-959, 1996).
A growing number of GPCRs with ligand independent activity exist whose biological function is most likely regulated at the transcriptional level (Leurs et al.,
Trends Biochem. Sci
. 23:418-422, 1998). While members of this class of GPCR are also subject to phosphorylation dependent downmodulation, transcriptional control of receptor number confers a high degree of temporal control over receptor turnover and activity.
Multiple GPCRs in a given cell-type can couple to the same G protein and a variety of accessory molecules exist which can modify both the responsiveness of GPCRs to effector signals (McLatchie et al.,
Nature
393:333-339, 1998) as well as integrating GTP exchange on G&agr; subunits with parallel signal transduction pathways to modify the biological outcome (Kehrl,
Immunity
8:1-10, 1998; Strittmatter et al.,
Nature
344:836-841, 1990). Nevertheless, biological/biochemical responses to activation of a GPCR are determined primarily by the nature of the G&agr; subunits to which it is coupled, and this cannot be predicted by primary sequence analysis of newly discovered GPCRs (Hedin et al.,
Cell Signal
5:505-518, 1993). A primary objective, therefore, in the study of an orphan GPCR is to define its G&agr; coupling profile. While direct experimental approaches such as photolabelling of G&agr; subunits with radiolabeled GTP analogues have been useful in studies of GPCRs with known ligands or agonists (Offermanns et al.,
Meth. Enzymol
. 195:286-301, 1991), they are limited in their application to the study of GPCRs in the absence of a defined ligand/agonist. However, signaling events downstream of many G&agr; subunits have been well defined and their analysis can serve as surrogate assays of G&agr; coupling profiles. Indeed, the biochemical/signaling properties of GPCRs are most often recapitulated in heterologous cell-types (Beadling et al.,
J. Immunol
. 162:2677-2682, 1999) and cell lines in which the spectrum of expressed G&agr; subunits are defined can serve as systems in which to study the primary signal transduction and biological characteristics of orphan GPCRs. Such approaches have therefore been used for preliminary analyses of orphan GPCRs as well as for ligand/drug screening protocols (Fraser,
J. Nucl. Med
. 36:17S-21S, 1995).
Interest in this family of receptors has increased with the realization of their potential clinical applications as drug targets. From the perspective of their clinical significance there is considerable focus on GPCRs expressed in the hematopoietic and lymphoid systems as many have been shown to play pivotal roles in the regulation of hematopoiesis and immune function. Receptor/ligand relationships within the GPCR family exhibit significant promiscuity, with many receptors recognizing more than one ligand and vice versa. This is especially true among chemokine receptors and a major goal, therefore, is to define the spectrum of receptor/ligand interactions within this family of GPCRs, which includes a number of lymphoid expressed orphan receptors of unknown function.
Interestingly, GPCRs have functional homologues in human cytomegalovirus and herpesvirus, suggesting that GPCRs may have been acquired during evolution for viral pathogenesis (Strader et al.,
FASEB J
., 9:745-754, 1995; Arvanitakis et al.
Nature
, 385:347-350, 1997; Murphy,
Annu. Rev. Immunol
. 12:593-633, 1994).
The importance of G protein-coupled receptors is further highlighted by the recent discoveries that its family members, chemokine receptors CXCR4/Fusin and CCR5, are co-receptors for T cell-tropic and macrophage-tropic HIV virus strains respectively (Alkhatib et al.,
Science
, 272:1955, 1996; Choe et al.,
Cell
, 85:1135, 1996; Deng et al.,
Nature
, 381:661, 1996; Doranz et al.,
Cell
, 85:1149, 1996; Dragic et al.,
Nature
, 381:667 (1996); Feng et al.,
Science
272:872, 1996). It is conceivable that blocking these receptors may prevent infection by the human immunodeficiency (HIV) virus.
Cell cycle checkpoints, intervals in the cell cycle in which the cell detects impairment or loss of integrity to its genome and arrests growth in order to make repairs, ensure that DNA is replicated with high fidelity (Paulovich et al.,
Cell
88:315-321, 1997; Hartwell,
Cell
71:543-546, 1992). There are three separately defined times in the eukaryotic cell cycle identified as checkpoints: G1/S transition, S-phase delay and G2/M transition (Nurse,
Cell
91:865-867, 1997). The G1/S checkpoint is activated to avoid copying mutated DNA by increasing the time available for repair. Cells also utilize a DNA damage checkpoint within S phase by slowing the rate of DNA replication. The G2/M checkpoint is activated upon detection of double-stranded DNA breaks. In addition, mitotic entry is monitored by a spindle checkpoint that inhibits anaphase progression when chromosomes are not attached to the mitotic spindle (Nicklas,
Science
275:632-637, 1997). The cell cycle checkpoint is summarized in FIG.
1
.
Recent discoveries have shed light on the molecular participants in the G2/M transition. Cdc2 and Cyclin B1 promote entry into mitosis and are part of the maturation promoting factor (MPF). Dephosphorylation of Cdc2 on Thr14 and Tyr15 by Cdc25 and phosphorylation on Thr161 concomitant with nuclear association with Cyclin B1 results in rapid entry into mitosis. Cyclin B1 degradation or export to the cytoplasm and phosphorylation of Cdc2 on the negative regulatory sites Thr14 and Tyr15 by Wee1 block entry into mitosis. Caffeine can relieve DNA damage-activated G2/M arrest by stimulating the dephosphorylation of Cdc2. These data strongly implicate MPF as the central regulator of the transition from G2 into mitosis.
Recent work has broadened our understanding of the signaling pathways involved in G2/M
Weng Zhigang
Witte Owen N.
Basi Nirmal S
Eyler Yvonne
The Regents of The University of California
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