Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Blood proteins or globulins – e.g. – proteoglycans – platelet...
Reexamination Certificate
2000-05-26
2003-01-21
Kunz, Gary L. (Department: 1647)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Blood proteins or globulins, e.g., proteoglycans, platelet...
C435S069900, C435S070100, C435S071100, C435S471000, C435S069100, C435S007200, C435S254200, C536S023100, C536S023500
Reexamination Certificate
active
06509447
ABSTRACT:
The present invention relates to chimeric G proteins, expression constructs therefor, yeast cells expressing such constructs and methods of making and using them.
G protein-coupled receptors are integral membrane proteins, characteristically with 7 transmembrane domains, which convey hormonal and sensory signals to the cell interior (see (1) for review). These receptors are commonly referred to as 7TM receptors or 7TMRs. The receptors respond to ligand binding by activating heterotrimeric G proteins composed of &agr;, &bgr; and &ggr; subunits. The G
&agr;
subunit is bound to GDP in the G protein trimer, and interaction with an activated (ligand bound) receptor induces the replacement of GDP with GTP. Accompanying conformational changes result in the dissociation of G
&agr;
-GTP and the G
&bgr;
/G
&ggr;
particle, either of which can modulate ion channel or enzyme effectors to cause signal propagation. The signal persists until G
&agr;
, which has GTPase activity, hydrolyses the bound GTP, allowing reassembly of the heterotrimer. Members of the RGS (Regulator of G protein Signalling) protein family regulate signal duration by acting as GAPs (GTPase activating proteins) for the G
&agr;
subunit (38) (17). The G protein signalling system appears to be common to all eukaryotes.
A well-characterised example of the trimeric G protein signalling system is the pheromone response pathway of the budding yeast Saccharomyces cerevisiae (20). Cells of the MATa mating type express a receptor encoded by the STE2 gene. This receptor becomes activated upon binding of the &agr;-factor mating pheromone, a peptide secreted by cells of the opposite (MAT&agr;) mating type. The yeast G protein is assembled from the products of the GPA1 (G
&agr;
), STE4 (G
&bgr;
), and STE18 (G
&ggr;
) genes. The G
&bgr;
/G
&ggr;
(Ste4p/Ste18p) particle released upon activation of the Ste2p receptor conveys the signal to a mitogen-activated protein kinase (MAPK) module. This leads to activation of the cyclin-dependent kinase inhibitor Far1p, causing cell cycle arrest and transcriptional induction of a set of genes involved in the mating process, including FUS1. The pathway is desensitised by Sst2p, a member of the RGS family. Cells of the opposite mating type (MAT&agr;) express a different receptor (Ste3p) and thereby respond to the pheromone (a-factor) secreted by MATa cells; otherwise the signalling apparatus utilised in the two mating types is the same.
At present, at least 16 G
&agr;
subunits, 5 G
&bgr;
subunits and 11 G
&ggr;
subunits have been identified in mammals, which can assemble a wide diversity of trimeric G proteins. On the basis of sequence homology, the G
&agr;
subunits fall into at least four families, related to G
&agr;i
, G
&agr;s
, G
&agr;q
, or G
&agr;12
. Typically, a given 7TM receptor activates only a single or small subset of G
&agr;
subunits. Thus even in cells which express multiple G
&agr;
subunits, signalling may be specific to particular G proteins and their downstream signalling pathways. A combination of approaches has defined several specific regions of the G
&agr;
subunit as key determinants of receptor/G protein specificity. These include regions in the N-terminus, the switch II to switch III regions (21) which are primarily responsible for binding G
&bgr;
/G
&ggr;
, and particularly regions of the C-terminus. For example, a cluster of mutations occurring in the C-terminal region of G
&agr;
have been found to cause defects in receptor coupling ((29)(13) and references therein). Also, peptides modelled on the C-termini of G
&agr;t
(transducin) and G
&agr;i2
compete for binding to rhodopsin and the &bgr;-adrenergic receptor, respectively (9)(30)(35) and antibodies directed against the G
&agr;
C-terminus can also block interaction with receptors (6)(10)(36). The normal response to agonist stimulation of the adenosine A
1
receptor in cultured mammalian cells (e.g. COS cells) is the activation of G
&agr;i
-family proteins, resulting in inhibition of adenylate cyclase. G
&agr;q
-containing heterotrimers in contrast do not normally respond to A
1
activation. However, G
&agr;q
subunit can be induced to interact with the A
1
receptor by exchanging the C-terminal four amino acids of G
&agr;q
for the corresponding amino acids of G
&agr;i2
(7). Hence, phospholipase C (an effector of activated G
&agr;q
) can be activated by adenosine A1 receptor agonists when signalling is mediated by a chimeric G
&agr;
subunit (7). The somatostatin SST
3
receptor is incompatible with G
&agr;s
, but can be coupled to the activation of adenylate cyclase in COS cells by replacement of five C-terminal amino acids of G
&agr;s
with corresponding residues from either G
&agr;i2
, which is known to interact with the SST
3
receptor, or from G
&agr;16
(18). G
&agr;15
and G
&agr;16
interact with a wide range of 7TM receptors (26), and are unusual in this respect. In crystal structures which have been solved for G
i1
(41) and G
t
(19) trimers, the G
&agr;
C-terminal tail lies on a flat, largely uncharged surface of the G protein trimer which also includes two lipid groups covalently attached to the G
&agr;
N-terminus and the G
&ggr;
C-terminus. This surface is believed to face the membrane and to be involved in interactions with the intracellular loops of the 7TM receptor.
Several reports have demonstrated that the yeast G protein signalling system can be coupled to heterologously expressed mammalian G protein-coupled (7TM) receptors. Some receptors, including the rat somatostatin SST
2
receptor (33), and the rat adenosine A
2a
receptor (34), can interact directly with the yeast G
&agr;
protein Gpa1p, whereas other receptors, including the human growth hormone releasing hormone receptor (GHRHR)(12), are incompatible with Gpa1p. In order to allow coupling of these receptors, the yeast G
&agr;
subunit can be deleted and the heterologous receptor co-expressed with a full-length mammalian G
&agr;
subunit. Alternatively, chimeric G
&agr;
subunits have been used in which the C-terminal domain (approx. one third of the peptide sequence) of Gpa1p is replaced with the equivalent region of a mammalian G
&agr;
subunit. See WO95/21925 (American Cyanamid Company) for both approaches. Chimeras, or other modified or heterologous G
&agr;
subunits must satisfy several criteria to be useful in a yeast coupling system. Most importantly, they must bind efficiently to yeast G
&bgr;
/G
&ggr;
to prevent signalling in the absence of activated 7TM receptor, and they must effectively interact with agonist-bound activated receptors to be capable of signal propagation.
Such heterologous or chimeric G
&agr;
components can facilitate the coupling of a heterologous 7TM receptor to the yeast signalling system (the pheromone response pathway) so that the effects of ligands such as potential new drug molecules on the receptor can be observed in phenotypic responses of the yeast cells. For example, cells may be induced to grow, and/or to convert an indicator dye compound in response to receptor agonists by introducing reporter genes downstream in the signalling pathway. However, chimeric constructs such as those described in WO95/21925 commonly show reduced affinity for the yeast G protein &bgr;&ggr; subunit pair and show increased background signalling. Because of the specificity of a given receptor for one or a small number of the known G
&agr;
subunits, different G
&agr;
constructs have been required to demonstrate functional coupling activity with the majority of receptors tested.
The discovery of new drugs able to act via 7TM receptors requires screens of high efficiency, yet high specificity. Currently, the yeast coupling system is constrained by receptor/G protein specificity which imposes a requirement for a wide variety of G
&agr;
constructs to be tried for each receptor tested for coupling. For receptors which fail to couple, there is uncertainty as to whether failure is due to receptor/G protein incompatibility, or other reasons such as inappropriate receptor conformation, for example. It would be desirab
Brown Andrew James
Dowell Simon Jeremy
Glaxo Welcome Inc.
Kunz Gary L.
Landsman Robert S.
Nixon & Vanderhye P.C.
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