Chemistry: molecular biology and microbiology – Treatment of micro-organisms or enzymes with electrical or... – Modification of viruses
Reexamination Certificate
1998-02-27
2001-07-03
Ulm, John (Department: 1646)
Chemistry: molecular biology and microbiology
Treatment of micro-organisms or enzymes with electrical or...
Modification of viruses
C435S007100, C435S007200, C436S501000, C530S350000, C536S023500
Reexamination Certificate
active
06255089
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates generally to the field of transmembrane receptors, more particularly to seven segment transmembrane G protein-coupled receptors, and most particularly to the serotonin (5-HT) receptors. Through genetic mutational techniques, the amino acid sequences of the native 5-HT
2A
and 5-HT
2C
receptors have been modified so that the receptors exist in a constitutively activated state exhibiting both a greater response to agonists and a coupling to the G Protein second messenger system even in the absence of agonist. A method for constitutively activating G protein-coupled 5-HT receptors in general is also disclosed.
2. Description of Related Art
The research interest in G protein-coupled cell surface receptors has exploded in recent years as it has been apparent that variants of these receptors play a significant role in the etiology of many severe human diseases. These receptors serve a diverse array of signalling pathways in a wide variety of cells and tissue types. Indeed, over the past 20 years, G protein-coupled receptors have proven to be excellent therapeutic targets with the development of several hundred drugs directed towards activating or deactivating them.
G protein-coupled receptors form a superfamily of receptors which are related both in their structure and their function. Structurally the receptors are large macromolecular proteins embedded in and spanning the cell membrane of the receiving cell and are distinguished by a common structural motif. All the receptors have seven domains of between 22 to 24 hydrophobic amino acids forming seven &agr; helixes arranged in a bundle which span the cell membrane substantially perpendicular to the cell membrane. The transmembrane helixes are joined by chains of hydrophilic amino acids. The amino terminal and three connecting chains extend into the extracellular environment while the carboxy terminal and three connecting chains extend into the intracellular environment. Signalling molecules are believed to be recognized by the parts of the receptor which span the membrane or lie on or above the extracellular surface of the cell membrane. The third intracellular loop joining helixes five and six is thought to be the most crucial domain involved in receptor/G protein coupling and responsible for the receptor selectivity for specific types of G proteins.
Functionally, all the receptors transmit the signal of an externally bound signalling molecule across the cell membrane to activate a heterotrimeric transducing protein which binds GDP (guanosine diphosphate). Upon activation, the bound GDP is converted to GTP (guanosine triphosphate). The activated G protein complex then triggers further intracellular biochemical activity. Different G proteins mediate different intracellular activities through various second messenger systems including, for example, 3′5′-cyclic AMP (cAMP), 3′5′-cyclic GMP (cGMP), 1,2-diacylglycerol, inositol 1,4,5-triphosphate, and Ca
2+
. Within the human genome, several hundred G protein-coupled receptors have been identified and endogenous ligands are known for approximately 100 of the group. While the seven transmembrane motif is common among the known receptors, the amino acid sequences vary considerably, with the most conserved regions consisting of the transmembrane helixes.
Binding of a signalling molecule to a G protein-coupled receptor is believed to alter the conformation of the receptor, and it is this conformational change which is thought responsible for the activation of the G protein. Accordingly, G protein-coupled receptors are thought to exist in the cell membrane in equilibrium between two states or conformations: an “inactive” state and an “active” state. In the “inactive” state (conformation) the receptor is unable to link to the intracellular transduction pathway and no biological response is produced. In the altered conformation, or “active” state, the receptor is able to link to the intracellular pathway to produce a biological response. Signalling molecules specific to the receptor are believed to produce a biological response by stabilizing the receptor in the active state.
Discoveries over the past several years have shown that G protein-coupled receptors can also be stabilized in the active conformation by means other than binding with the appropriate signal molecule. Four principal methods have been identified: 1) molecular alterations in the amino acid sequence at specific sites; 2) stimulation with anti-peptide antibodies; 3) over-expression in in vitro systems; and 4) over-expression of the coupling G proteins. These other means simulate the stabilizing effect of the signalling molecule to keep the receptor in the active, coupled, state. Such stabilization in the active state is termed “constitutive receptor activation”.
Several features distinguish the constitutively activated receptors. First, they have an affinity for the native signalling molecule and related agonists which is typically greater than that of the native receptors. Second, where several known agonists of varying activity (to the native receptor) were known, it was found that the greater the initial activity of the agonist, the greater was the increase in its affinity for the constitutively activated receptor. Third, the affinity of the constitutively activated receptor for antagonists is not increased over the affinity for the antagonist of the native receptor. Fourth, the constitutively activated receptors remain coupled to the second messenger pathway and produce a biological response even in the absence of the signalling molecule or other agonist.
The importance of constitutively activated receptors to biological research and drug discovery cannot be overstated. First, these receptors provide an opportunity to study the structure of the active state and provide insights into how the receptor is controlled and the steps in receptor activation. Second, the constitutively active receptors allow study of the mechanisms by which coupling to G proteins is achieved as well as how G protein specificity is determined. Third, mutated constitutively active receptors are now recognized in disease states. Study of constitutively activated receptors has demonstrated that many mutations may lead to constitutive activation and that a whole range of activation is possible. Fourth, the existence of constitutively active receptors provides a novel screening mechanism with which compounds which act to increase or decrease receptor activity can be identified and evaluated. Such compounds may become lead compounds for drug research. Finally, studying the affect of classical antagonists (compounds previously identified as, in the absence of agonist, binding to the receptor but causing no change in receptor activitiy, and, in the presence of agonist, competitively decreasing the activity of a receptor) and other drugs used as treatments on the constitutively active receptors has led to the discovery that there are compounds, inverse agonists, which decrease the constitutive activity of the active state of the receptors but which have no or little affect on the inactive state. The difference between antagonists, which act on the inactive state, and inverse agonists, which act on the active state, is only discernable when the receptor exhibits constitutive activity. These inverse agonists, identifiable with constitutively active receptors, present an entirely new class of potential compounds for drug discovery.
About 10 years ago, it was recognized that neurotransmitter receptors can be divided into two general classes depending on the rapidity of their response. Fast receptors were identified with ion channels and mediate millisecond responses while slower receptors were identified with G protein-coupled receptors. These G protein-coupled receptors include certain subtypes of the adrenergic as well as the muscarinic cholinergic (M1-M5), dopaminergic (D1-D5), serotonergic (5-HT1, 5-HT2, 5-HT4-5-HT7) and opiate (&dgr;, &kgr;, an
Egan Christina C.
Herrick-Davis Katharine
Teitler Milt
Albany Medical College
Burgoon, Jr. Richard P.
Nguyen Ann A.
Ulm John
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