Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving transferase
Reexamination Certificate
2000-06-02
2003-06-03
Prouty, Rebecca E. (Department: 1652)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving transferase
C536S023200, C536S023400, C435S069100, C435S194000, C435S252300, C435S320100, C435S325000, C435S069700
Reexamination Certificate
active
06573059
ABSTRACT:
FIELD OF THE INVENTION
The invention is directed to the measurement of cAMP either in vitro or within living cells. The invention further relates to molecules for use in the methods and to DNA constructs that encode the molecules.
BACKGROUND OF THE INVENTION
In molecular biology, drug testing and medical diagnostic, it is desirable to measure cAMP concentration, since intracellular cAMP is a common second messenger in many living cells. cAMP concentration in solution is currently routinely measured using either heart muscle protein extracts or antibodies (Amersham, cAMP assays, TRK432 or RPA509).
In order to measure cAMP levels, the cells need to be lysed and cAMP solubilized. Such methods require time and are prone to artifacts resulting from cAMP degradation by phosphodiesterases, incomplete lysis or masking agents. Current methods based on muscle extracts allow to measure concentrations of solubilized cAMP in the range of 0.125-32 pmol/ml and radioimmunoassay 0.25-16 pmol/ml. Acetylation allows to improve these detection limits by factors of 3 to 10. Scintillation proximity assays further simplified radioimmunoassays (Amersham, RPA538) without much improving detection thresholds.
There remains a need for better cAMP measurement methods that can be used in vitro and in vivo.
The present invention provides such methods and is based on the following observations.
The regulatory subunit of the cAMP dependent protein kinase (PKA) from Dictyostelium shows a K
D
for cAMP of about 20 nM, which is in the same range as mammalian enzymes 10-30 nM. It was considered that this PKA could thus represent an alternative system for the measurement of cAMP.
Furthermore, this protein can be produced within any cell by placing its gene in a proper expression vector, thus also allowing intracellular cAMP measurements.
Binding of extracellular signals, like hormones, to membrane bound receptors triggers an increase in cAMP concentration within the cell. Intracellular cAMP binds mainly to the regulatory subunit of PKA, dissociating regulatory (R) and catalytic (C) subunits. The liberated catalytic subunit is then able to phosphorylate numerous substrates, ranging from enzymes regulating metabolic pathways to transcription factors. Measurement of intracellular cAMP concentration thus reflects the activation state of a particular cell after an external stimulus. A way to trace intracellular cAMP increase has been fluorescence ratio imaging to monitor the proportion and localisation of R-C complexes (Adams et al., 1991, Nature 349, 694-697). However, the need for labelling the proteins with fluorophores in vitro and their subsequent reintroduction within calls by microinjection prevented generalisation of this method.
The cAMP dependent protein kinase (PKA) is almost ubiquitous in eukaryotic cells. In mammals PKA is composed of an haterotetramer made of two regulatory (R) and two catalytic subunits (C) which are encoded by different genes. In Dictyostelium, PKA forms only a heterodimer with one R and one C subunit. The R subunit from Dictyostelium resembles closely the mammalian RII type and can interact with mammalian C subunits (Reymond and Veron, 1995, DdPKA, cAMP-dependent PK (
D. discoideum
) In: The protein kinase Facts book, protein-serine kinases, G. Hardie and S. Hanks, eds. (London: Academic Press), pp. 70-72). However the Dictyostelium R subunit has the unique property of not forming a dimer naturally. The inventors anticipated that this should facilitate cAMP binding as well as R-C interaction studies.
There is however a need for a reporter molecule within living cells which could detect cAMP changes.
The isolation of a gene encoding a green fluorescent protein (GFP) from
Aequorea victoria
opened a new way to monitor proteins within cells non-invasively using the techniques of fluorescence microscopy or flow cytometry (Chalfie et al, 1994, Science 263, 802-805, and U.S. Pat. No. 5,491,084). The GFP protein undergoes an autocatalytic reaction involving Ser65, Tyr66 and Gly67 residues, leading to the creation of a fluorophore. A series of mutations have been introduced around amino acid 66, allowing to modify both excitation and emission wavelength (U.S. Pat. No. 5,777,079).
The use of two GFPs acting as donor and acceptor fluorophores has allowed to obtain fluorescence energy transfer (FRET). When excited at the proper wavelength, the donor GFP emits light in the range of the excitation wavelength of the acceptor GFP. FRET depends on the distance (d) between the fluorophores and decreases as a function of d
6
, thus donor and acceptor GFPs have to be placed in close proximity. As a result of FRET, the donor emission peak is reduced, while the acceptor emission increases.
A major limitation of the use of GFPs, however, has been the insertion of the GFPs either at the N- or C-terminus of the protein of interest. This type of insertion results in many cares in an inactivation of the protein of interest.
In the research that led to the present invention it has been found that GFPs can be inserted at almost any position within the R-subunit without loosing its ability to fluoresce (Biondi et al., 1998, Nucleic Acids Research 26, 4946-52). Furthermore, functional R-subunit properties, namely cAMP binding and interaction with the C-subunit, were kept in many fusions. It was anticipated that such proteins can be used to monitor cAMP binding. However, one would prefer a test in which fluorescence changes upon cAMP binding.
According to the present invention it was now demonstrated that particular R-GFP fusions from
Dictyostelium discoideum
can be used for cAMP measurements, based on FRET changes. These R-GFP fusions can be used also within living cells. In addition, a truncated R subunit, able to bind a single cAMP molecule with high affinity, is further used to obtain simple quantification. The invention thus provides a cAMP binding protein that is modified by fusion to fluorescent proteins.
More in particular, the present invention provides DNA constructs and methods allowing to monitor by fluorescence changes the binding of cAMP to the regulatory subunit of the cAMP dependent protein kinase (PKA) from
Dictyostelium discoideum
. This method in nature is applicable both to in vitro and in vivo tests, since the gene encoding the R subunit, as well as fusions with green fluorescent proteins (GFP) can be expressed in different organisms ranging from bacteria to human cells.
In the present invention, methods and compositions are provided for producing Dictyostelium R-subunits in
E. coli
. DNA constructs allowing the expression of fusion proteins in
E. coli
are described in which donor and/or acceptor GFPs are inserted at particular locations within the R subunit allowing fluorescence energy transfer (FRET). Evidences for the occurrence of FRET are presented either between fluorescent cAMP or cGMP, or between acceptor and donor GFPs. FRET in modified upon cAMP binding Thus the GFP-R fusion proteins presented can be applied for the measurement of cAMP concentration.
Furthermore, the nature of the fused genes is compatible with expression in living cells, allowing to measure intracellular cAMP concentrations in vivo.
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Biondi et al., “Random insertion of GRP into the cAMP-dependent protein kinase regulatory subunit from Dictyostelium discoideum,” Nucleic Acids Research., vol. 26, No. 21, Nov. 1, 1998, pp. 4946-4952.
Mucignat-Caretta et al., “Binding of two fluorescent cAMP analogues to type I and II regulatory subunits of cAMP-dependent protein kinases,” Biochim Biophys Acta., vol. 1357, No. 1, Jun. 5, 1997, pp. 81-90.
Pollock et al., “Using GFP in FRET-based applications,” Trends Cell Biol., vol. 9, No. 2, Feb. 1999, pp. 57-60.
Adams et al., “Fluorescence ratio imaging of cy
Pillsbury & Winthrop LLP
Prouty Rebecca E.
Ramirez Delia
RMF Dictagene S.A.
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