Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
1995-06-06
2001-07-03
Ulm, John (Department: 1646)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S252300, C435S320100, C536S023200
Reexamination Certificate
active
06255069
ABSTRACT:
BACKGROUND OF THE INVENTION
G protein-coupled receptors represent a diverse family of cell surface proteins that transduce the binding of extracellular ligands (hormones, neurotransmitters, odorants, light, etc.) into intracellular signalling events. G protein-coupled receptors modulate the activity of a wide variety of effector molecules including adenylyl cyclase, cGMP phosphodiesterase, phospholipase C, phospholipase A2, and K
+
, Na
+
and Ca
++
channels. It has become apparent in recent years that G protein-coupled receptor kinases play a vital role in regulating receptor function by their unique ability to specifically phosphorylate activated forms of G protein-coupled receptors.
Two of the best characterized G protein-coupled receptors are the hormone responsive &bgr;
2
-adrenergic receptor (&bgr;
2
AR), which mediates catecholamine stimulation of adenylyl cyclase, and the visual “light receptor” rhodopsin, which mediates phototransduction in retinal rod cells. The &bgr;
2
AR and rhodopsin share many structural and functional similarities including a conserved protein topology (e.g., seven transmembrane domains) as well as an ability to specifically interact with G proteins upon activation. The similarities between these receptors also extend to mechanisms involved in receptor regulation. In both of these systems, a rapid loss of receptor responsiveness occurs following activation. This rapid activation-dependent loss of responsiveness or desensitization is promoted by phosphorylation of the receptor. This phosphorylation is mediated by protein kinases that have the unique ability to recognize and phosphorylate their receptor substrates only when they are in their active conformations, i.e., when they have been stimulated and/or occupied by appropriate agonist ligands. The &bgr;-adrenergic receptor kinase (&bgr;ARK) and rhodopsin kinase have been identified as kinases involved in the agonist-specific phosphorylation of the &bgr;
2
AR and rhodopsin, respectively. The subsequent uncoupling of the receptor and G protein is then mediated by arrestin proteins that specifically bind to the phosphorylated and activated form of the receptor. Additional lines of evidence suggest that other G protein-coupled receptors may also be regulated by similar mechanisms. These receptors include the m2 muscarinic cholinergic and &agr;2-adrenergic receptors, which inhibit adenylyl cyclase; the &agr; mating factor receptor of the yeast
Saccharomyces cerevisiae
and the chemotactic cAMP receptor of the slime mold
Dictyostelium discoideum.
Several lines of evidence suggest that &bgr;ARK may have a broad substrate specificity and thus serve as a general G protein-coupled receptor kinase. First, direct phosphorylation studies have demonstrated that &bgr;ARK not only phosphorylates the &bgr;
2
AR to a high stoichiometry, but also can phosphorylate the &agr;2-adrenergic, the m2 muscarinic cholinergic and the substance P receptors. In addition, several agents appear to promote an increase in membrane-associated &bgr;ARK activity. These include &bgr;-agonists, prostaglandin E1, somatostatin, and platelet activating factor, suggesting the involvement of &bgr;ARK in the regulation of these receptors. Moreover, a growing body of evidence suggests that &bgr;ARK and rhodopsin kinase are members of a multigene family.
Structural information on the G protein-coupled receptor kinase (GRK) family was initially provided by the isolation of a cDNA encoding bovine &bgr;ARK [Benovic, J. L., DeBlasi, A., Stone, W. C., Caron, M. G., and Lefkowitz, R. J., 1989
Science,
246:235-246]. &bgr;ARK is a protein of 689 amino acids (79.6 kD) containing a central protein kinase catalytic domain flanked by large amino and carboxyl terminal domains. Additional members of the GRK family were subsequently cloned including bovine &bgr;ARK2 [Benovic, J. L., Onorato, J. J., Ariza, J. L., Stone, W. C., Lohse, M., Jenkins, N. A., Gilbert, D. J., Copeland, N. G., Caron, M. G. and Lefkowitz, R. J., 1991
J. Biol. Chem.,
266:14939-14946], bovine rhodopsin kinase [Lorenz, W., Inglese, J., Palczewski, K., Onorato, J. J., Caron, M. G. andLefkowitz, R. J., 1991
Proc. Natl. Acad. Sci. USA,
88:8715-8719], Drosophila kinases GPRK-1 and GPRK-2 [Cassill, J. A., Whitney, M., Joazeiro, C. A. P., Becker, A., and Zucker, C. S., 1991
Proc. Natl. Acad. Sci. USA,
88:11067-11070], and the recently identified human IT11 [Ambrose, C., James, M., Barnes, G., Lin, C., Bates, G., Altherr, M., Duyao, M., Groot, N., Church, D., Wasmuth, J. J., Lehrach, H., Housman, D., Buckler, A., Gusella, J. F., and MacDonald, M. E., 1992
Human Mol. Genet.,
1:697-703]. &bgr;ARK2 and Drosophila GPRK-1 appear to be the most similar to &bgr;ARK with amino acid identities of 84% and 64%, respectively. In contrast, rhodopsin kinase, IT11, and Drosophila GPRK-2 have significantly lower homology with &bgr;ARK (35-406 amino acid identity). Common features of these kinases include a centrally localized catalytic domain of approximately 240 amino acids which shares significant amino acid identity (46 to 95%), an N-terminal domain of 161-197 amino acids (except for GPRK-2), and a variable length C-terminal domain of 100-263 amino acids.
The tremendous diversity in the G protein-coupled receptor family suggested that there may well be other GRK members. This has, in fact, been found to be the case. In the present invention, two additional members of the GRK gene family have been identified, GRK5 and GRK6; these cDNAs have now been cloned, expressed and characterized.
SUMMARY OF THE INVENTION
G protein-coupled receptor kinases (GRK) play an important role in phosphorylating and regulating the activity of G protein-coupled receptors. Complementary DNAs (cDNAs) that encode two novel members of the G protein-coupled receptor kinase family are provided in the present invention. These cDNAS (SEQ ID NO: 1 and SEQ Id NO: 2, respectively) encode GRK5 (590 amino acids; SEQ ID NO:5) and GRK6 (576 amino acids; SEQ ID NO:6) which represent two new members of the GRK family that have distinct tissue distribution and substrate specificity. These kinases have been overexpressed in Sf9 insect cells, purified and characterized. Using the purified kinases, it has been demonstrated that heparin is a potent inhibitor of GRK5 (IC
50
~1 nM) and a good inhibitor of GRK6 (IC
50
~15 nM). These results indicate that specific inhibitors of these two kinases can be identified.
The GRK5 and GRK6 cDNAS can be expressed, purified and further characterized with regard to their substrate specificity. The availability of the cDNAs will also enable the generation of reagents to block the activity of the endogenous kinases. These include dominant negative mutations which involves mutating a critical lysine residue in the kinase to arginine. This results in a protein kinase which can still bind to its receptor substrate but can no longer transfer a phosphate group to the receptor. Thus, the mutant kinase can block the ability of the endogenous kinase to phosphorylate a given receptor and thereby block desensitization. An alternative strategy for blocking the activity of the endogenous kinase involves the use of antisense oligonucleotides or stably transfected antisense constructs to block expression of the kinase. This will generate a cell with a reduced ability to desensitize to various agents. Expression of GRK5 and GRK6 will also allow screening for specific inhibitors of these two kinases. It has been demonstrated that heparin is a potent inhibitor of GRK5 and a good inhibitor of GRK6. Inhibitors are identified and then utilized to specifically block the activity of GRK5 or GRK6, and thus inhibit receptor desensitization. Specific inhibitors of these kinases may be used therapeutically to either directly modulate the activity of a given receptor (by blocking endogenous desensitization of that receptor) or by augmenting the ability of a given therapeutic agent to stimulate a given receptor (again by blocking desensitization). Alternativel
Benovic Jeffrey L.
Gomez Jorge
Kunapuli Priya
Kumar Nanda P. B. A.
McNichol, Jr. William J.
ReedSmith LLP
Thomas Jefferson University
Ulm John
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