The nature has its own solutions for communications among trillions of cells that are remotely located through out the living system. The cells being unit of life and nucleus being the center, it needs to know what is happening through out the body so that it can dynamically respond to the situation in order to maintain homeostasis. The cell signalling and signal transduction is a fertile topic of research with fundamental and applied therapeutic point of view. The signals ultimately should reach nucleus and responses should arise from it. In a cell, as in human body like brain, nucleus is also not accessible directly, as it is located deeply inside the cytoplasm.
GPCRs are one of the most important drug targets for the pharmaceutical industry, and more than 30% of all marketed therapeutics act on them. However, these drugs target only approximately 30 members of the family, mainly biogenic amine receptors, so there is enormous potential within the pharmaceutical industry to exploit the remaining family members, including the more than 100 orphan receptors for which no existing ligands have so far been identified.
The cell receives and responds to variety of signals at boundary, which is usually the cell membrane. There are various proteins localized in the membrane which act as receptors of signals. GPCR (Guanine-protein coupled receptors) are the most prominent signal transducers. These are the receptors for biogenic amines, peptides, Acetylcholine, eicosanoids, Wnt proteins, adhesion proteins, odorants and photons. The effectors of these across the membrane involve increase/decrease of cAMP concentration by modulation of adenyl cyclase, activation of phosopholipase Cß or activation of P1-3 kinases through Gß? subunit and modulation of inward rectified potassium currents. Hundreds of cell-surface receptors for hormones and other ligands use 'G proteins' to transduce intracellular signalling pathways. Dramatic recent advances by several laboratories have provided exciting details of the structure of the Heterotrimeric G proteins. The full name of "G protein" is GTP-binding protein because in the active state it binds to GTP (guanosine triphosphate). There are two types of G proteins: Heterotrimeric G proteins and Monomeric G proteins (or small G proteins). G-protein-coupled receptors are coupled to Heterotrimeric G proteins. Heterotrimeric G proteins are key transducers for signal transfer from outside of the cell. The Heterotrimeric G protein consists of three subunits: a , ß and ?, Based on the differences in their genes, 20 a, 6 ß and 12 ? subunits have been identified. Their molecular weights are in the range 8 kD to 46 kD (a subunit: 39 - 46 kD; ß subunit: 35 - 39 kD ; ? ?subunit: ~ 8 kD ) . In the inactive state, the a subunit binds to GDP and the three subunits are attached together (see figure 1). When the a subunit binds to GTP, its affinity to the ß ? subunits is decreased, resulting in their dissociation. The separated a and/or ß ? subunits can then interact with their effectors. GPCR signalling path way involves every organ system and present a wide range of opportunities as therapeutic targets in areas including cancer, cardiac dysfunction, diabetes, central nervous system disorders, obesity, inflammation, and pain. Consequently, GPCRs are prominent components of pipelines in small and large drug companies alike, and many drug discovery firms focus exclusively on these receptor.
Accessory proteins may regulate the strength/efficiency/specificity of signal transfer from receptor to G protein or G protein to effector, help position these three core signaling components in the right microenvironment, and/or contribute to the formation of a functional signal transduction complex. Such a complex may exist in the absence of the stimuli or its formation may be initiated by receptor activation. The signal transduction network for this system may parallel that used by receptors with a single-membrane-span motif, where binding of agonist initiates a series of protein interactions that depend on protein phosphorylation.
Recently additional accessory proteins that influence guanine nucleotide binding and/or hydrolysis of subunit interactions, which also regulate many if not, all of the subtypes of Heterotrimeric G proteins have come to knowledge. Activators of G protein signaling (AGS) refer to a functionally defined group of proteins that activate G protein-signaling systems in the absence of a classical G protein-coupled receptor. AGS and related proteins provide unexpected insights into the regulation of the G protein activation/deactivation cycle and the functional roles of G proteins. These proteins are likely to play important roles in the generation of signaling complexes, the positioning of signaling proteins within the cell, and in biological roles of G proteins unrelated to a cell surface receptor. As such, these proteins and the concepts advanced with their discovery provide unexpected avenues for therapeutics and understanding mechanisms of diseases.
AGS, when initially discovered in 1998 were named as RAMPs (Receptor Activity-Modifying Proteins; for e.g., needed to the functional activity of calcitonin gene-related peptide). Three RAMPs generated by three different genes are known in human, rat and mice. The coding sequences of such genes are described, however the regulation sequences are yet unknown. GPCR comprises of variety of signalling molecules considered as super family of receptor. They are classified as class I (Retinoids) Class II (Calcitonin) and Class III. Alternatively they are classified on the basis of a subunit as as , ai , aq , ao atolf and a12/13 . RAMPs interact with GPCR of class II. However, the complex GPCR1/RAMP2 enhances specifically the phosphoinoside-signaling pathway. It is well established that AGS may provide a cell-specific mechanism for signal amplification by acting in concert with GPCRs. They may also influence the population of activated G protein/effectors within the cell independent of receptor activation. They may also be "effectors" subject to receptor regulation providing attractive targets for cross talk among diverse signaling systems. They may provide alternative modes of input to G protein-regulated signaling pathways independent of classical GPCRs. Such accessory proteins thus have potentially broad physiological and pharmacological significance relative to the cell biology and functional properties of G proteins themselves. By contributing to the amplification of biological stimuli commonly observed with signaling events involving Heterotrimeric G proteins, these proteins may be of particular importance in tissues requiring rapid signal processing or under conditions of aberrant cell growth. The modulation of key signaling pathways should present some interesting opportunities for drug development also. Agents that influence the activity of these accessory proteins may impact GPCR signaling by altering signal duration or intensity and perhaps modulate receptor regulatory mechanisms such as desensitization of GPCRs.
The finding of AGS has excited the pharmacologists worldwide as these can be novel targets for drug development. Intracellular accessory proteins can be critical for G protein-coupled receptor (GPCR) biogenesis, including aspects of GPCR trafficking. Recent discoveries include the identification of multiple membrane-associated proteins that dictate not only the intracellular sequestration and/or transport of GPCRs, but also modulate-quite dramatically-GPCR ligand specificity subsequent to delivery to the cell surface.
The identification of three novel receptor-activity-modifying proteins (RAMP) revealed a new functional principle of G protein-coupled receptors with seven transmembrane-domains. Calcitonin receptors (CTR) and calcitonin receptorlike receptors (CLR) of the B family of these receptors use RAMP as accessory proteins at the cell surface to modulate the specificity for the peptides of the calcitonin family such as of calcitonin generelated peptide (CGRP), adrenomedullin and amylin. The CTR/RAMP1 and the CLR/RAMP1 complexes are CGRP/amylin- and CGRP-receptors, respectively, distinguished by calcitonin and CGRP antagonists. Another amylin receptor isotype is revealed in cells coexpressing CTR and RAMP3. RAMP2 and -3 associate with the CLR defining adrenomedullin receptors. Thus, the CTR and the CLR individually interact with the three RAMP at the cell surface to form high affinity receptors for the four peptides of the calcitonin family. Differential actions of CGRP and adrenomedullin in the pulmonary and cardiovascular systems are studied in transgenic animals that overexpress corresponding receptors and mutants thereoff. Overexpression of the CLR in transgenic mice under control of a smooth muscle alpha-actin promoter revealed a glaucoma-like phenotype and skeletal defects.
Regulators of G protein signaling (RGS) are an important family of proteins that negatively modulate signaling through G protein-coupled receptors (GPCRs). Several members of this family are highly expressed in mesolimbic brain regions associated with reward; moreover, this expression changes in response to exposure to opioids, cocaine, and amphetamine, sometimes very rapidly.
There is growing evidence that drugs of abuse alter the expression of RGS proteins. These proteins modulate the rewarding effects that contribute to continual drug use. The mounting evidence suggests of a role for RGS9-2, as a negative regulator of the abuse liability properties of both opioids and stimulants. Consequently, pharmacological agents that target this protein might alter behavioral responses to opioids and stimulants. Nevertheless, cell signaling is highly complex, and there is a myriad of accessory proteins and protein-protein interactions whose precise functions in drug signaling have yet to be elucidated. For example, the mammalian RGS protein family has at least twenty members that can inhibit GPCR signaling. As we learn more about RGS proteins-especially about their various functional domains and their distribution and regulation-and other accessory proteins that modulate GPCR signaling, we should be able to identify particular RGS protein targets for medication development.
In view of recent findings, there is little doubt that GPCRs do not necessarily just exist and function as on interacting monomeric species or Heterodimers. The concept that a substantial range of GPCR and activators of GPCRs are expressed in native cells and tissues is beginning to encourage discussion as to how they might be best identified and whether they could provide novel and attractive. This is a rapidly expanding area of pharmaceutical research that holds great promise for delivering new and improved therapeutics in the near future.
The authors are with Birla Institute of Technology and Science, Pilani, 333031.
E mail: anantha@bits-pilani.ac.in