The nature has its own solutions for communications among trillions of cells that are remotely located throughout the living system. The cells being unit of life and nucleus being the centre, it needs to know what is happening throughout 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.
G protein-coupled receptors (GPCRs) are one of the most important drug targets for the pharmaceutical industry, and more than 30 per cent of all marketed therapeutics act on them.
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 b and g. Based on the differences in their genes, 20 a, 6 b and 12 g subunits have been identified. Their molecular weights are in the range of 8 kD to 46 kD (a subunit: 39 - 46 kD; b subunit: 35 - 39 kD; g subunit: ~ 8 kD). In the inactive state, the a subunit binds to GDP and the three subunits are attached together. When the a subunit binds to GTP, its affinity to the b g subunits decreases, resulting in their dissociation. The separated a and/or b g subunits can then interact with their effectors.
GPCR signalling pathway involves every organ system and presents 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 receptors.
Accessory proteins may regulate the strength/efficiency/ specificity of signal transfer from receptor to G protein or G protein to effector and help position these three core signalling 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 light. Activators of G protein signalling (AGS) refer to a functionally defined group of proteins that activate G protein-signalling 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, 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 receptor activity-modifying proteins (RAMPs). 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 signalling 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 signalling systems. They may provide alternative modes of input to G protein regulated signalling 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 signalling 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 signalling pathways should present some interesting opportunities for drug development also. Agents that influence the activity of these accessory proteins may impact GPCR signalling by altering signal duration or intensity and perhaps modulate receptor regulatory mechanisms such as desensitisation 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 receptor-like 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 gene-related 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 over express corresponding receptors and mutants thereof. Over expression 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 signalling (RGS) are an important family of proteins that negatively modulate signalling 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 behavioural 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 signalling have yet to be elucidated. For example, the mammalian RGS protein family has at least twenty members that can inhibit GPCR signalling.
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 prove 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)