Neuropeptides have been traditionally identified on the basis of three characteristics. Their abilities to modulate neurotransmitter release, most often acetylcholine, in organ assays has been used as a monitoring assay in the isolation of new neuropeptides. Substance P, the first neuropeptide discovered 75 years ago by Von Euler and Gaddum (1931), and the enkephalins discovered in 1975 by Hughes and Kosterlitz (Hughes et al., 1975) stand as forefront runners of this line of research. Neuropeptides have also been isolated on the basis of their chemical properties. Mutt and Tatemoto used the knowledge that some neuropeptides are C-terminally amidated to isolate new bioactive peptides that feature this chemical modification from large amount of intestine or brain tissues (Tatemoto and Mutt, 1980). Erspamer and Melchiorri took advantage of the fact that amphibian skin is an inexhaustible mine of active compounds to identify biologically active amines and bioactive peptides (Erspamer et al., 1978).
With the advent of molecular cloning, neuropeptides began to be also found on the basis of mRNA sequences. γ-Melanocyte stimulating hormone (γ-MSH) was found as a processing product of the proopiomelanocortin (POMC) precursor (Nakanishi et al., 1979) and calcitonin gene-related peptides (CGRP) was found as a product of the alternate processing of the calcitonin gene (Rosenfeld et al., 1983). More recently, genomic data searches taking advantage of structural features expected to be intrinsic to neuropeptides, such as being flanked by processing sites or exhibiting conserved sequences among different species, have also been used to pan out of the genome sequence potential new neuropeptides. Obestatin is the latest example of such a search (Zhang et al., 2005). By applying mRNA subtraction techniques, while searching for novel genes specifically expressed in hypothalamus, new potential neuropeptide precursors have been identified which led to the discovery of new neuropeptides, in particular orexins/hypocretins (Gautvik et al., 1996).
Except for the discoveries based on neurotransmitter release, the other approaches faced one challenge: how to insure that the newly found neuropeptides were genuine first messengers and not inactive products of the metabolism or processing of larger proteins. The cloning of receptors allowed devising an approach that overcomes this challenge. One of the fundamental characteristic of all neuropeptides is that they carry their message by interacting with G protein coupled receptors (GPCRs). An approach was developed which uses GPCRs as targets to isolate new neuropeptides. It combines the use of molecular cloning techniques with the traditional search for bioactivity in tissue extracts. It is by using this approach that most new neuropeptides have been discovered in the last 10 years. This approach is the subject of this review.
The G protein coupled receptors (GPCRs) form one of the largest gene superfamilies (Vassilatis et al., 2003) arose from the concept that receptors that couple to G protein to induce second messenger responses share similar structural features, hallmarked by their seven transmembrane topology. The GPCRs are activated by various signaling molecules such as monoamines, amino acid derivatives, peptides, lipids as well as chemoattractants, olfactants and light (Bockaert and Pin, 1999). They are central to intercellular interactions. Most notably, all the receptors that are activated by bioactive peptides, and in particular neuropeptides, are GPCRs.
The recognition that GPCRs share sequence similarities (Dixon et al., 1986) opened the door to the application of homology screening approaches to search for new ones. In 1980s and 1990s, low-stringency hybridization and PCR-based homology screening identify hundred of GPCRs (Bunzow et al., 1988, Libert et al., 1989). With the completion of the human genome sequences, the number of GPCRs has reached about 800 (Vassilatis et al., 2003). The identification of GPCRs on the basis of their sequence similarities, however, faces one major challenge: finding their endogenous ligands, i.e. determining their pharmacological nature. The GPCRs identified through this approach were all “orphan” GPCRs, receptors in search of their ligands. Indeed, the first reports of broad homology-based GPCR searches resulted in the identification of several orphan GPCRs (Bunzow et al., 1988, Libert et al., 1989). The solution to this challenge was to screen the orphan GPCRs against potential ligands, an approach that is referred to as reverse pharmacology (Civelli et al., 2001). Each orphan receptor is expressed in eukaryotic cells by DNA transfection, and the resulting cells are used as targets to determine which potential ligand bind to the orphan receptor either at the level of the membranes or by its ability to induce a second messenger response. And, already in 1988, the first deorphanized GPCRs were found to be the 5HT-1A and the D2 dopamine receptors (Bunzow et al., 1988, Fargin et al., 1988). By the mid 1990s, numerous research teams had joined this search and some 90 GPCRs had been matched to their respective natural ligands (Civelli et al., 2005).
But while this was on-going, we also came to the recognition that the number of GPCRs outscored the number of known ligands. This led us to propose that orphan GPCRs could be used as targets to identify novel endogenous ligands. An approach was devised aiming at finding endogenous ligands of orphan GPCRs not in ligand libraries but in tissues expected to produce them (Civelli et al., 2001). The strategy, sometimes referred to as the orphan receptor strategy, remains the same, the orphan GPCR is expressed in eukaryotic cells but these are tested against tissue extracts, often brain extracts since in the CNS the receptor and the secreted ligand are colocalized. Receptor reactivity is monitored by quantifying changes in second messenger levels. This approach faces two unknowns, the chemical nature of the ligand and the type of second messenger to be monitored.
Overcoming the first unknown relies mainly on the recognition that sequentially related GPCRs bind chemically related ligands. Therefore, an orphan GPCR that belongs phylogenetically to a particular family of GPCRs should bind a ligand chemically similar to the ligands that bind the other GPCRs of that family. The second unknown could not be answered by sequence analyses as there are no rules that link a GPCR sequence to a particular G protein. This unknown has often been overcome either by random testing, by taking advantage of the promiscuity that overexpressed GPCRs often exhibit for G protein coupling or by forcing GPCRs to a particular second messenger systems by coexpressing G protein chimera (Mody et al., 2000). All together, this approach requires the ability to carry out larger number of tests since tissue extracts must be fractionated before being tested and since different second messenger responses may have to be monitored.
The first success of the orphan receptor strategy was the discovery of a novel neuropeptide. An orphan GPCR, ORL-1, had been identified based on its homology to opioid receptors but shown not to be activated by any of endogenous opioid peptides or opiates (Mollereau et al., 1994). By applying the orphan receptor strategy, a 17 amino acid peptide was identified as the natural ligand of ORL1. It was named orphanin FQ or nociceptin (OFQ/N) and found to share some similarity to the opioid peptides, while specific for ORL-1 (Meunier et al., 1995, Reinscheid et al., 1995). It has since been shown to be involved in various physiological functions such as anxiety, pain, analgesia, memory as well as stress (Mogil and Pasternak, 2001). This success opened the door for the search of novel neuropeptides. Over the last decade, 13 novel neuropeptide families have been discovered using the orphan receptor strategy (Table 1). This review will discuss several of them focusing on the impact that they have on our understanding of two physiological responses, sleep and feeding.
In 1930, von Economo predicted that two discrete brain regions regulate sleep: a wake-promoting region in the posterior hypothalamus and a sleep-promoting region in the preoptic area (Von Economo, 1930). He identified them from patients with encephalitis lethargica presumably caused by a viral infection during World War I. These patients had either prolonged sleepiness caused by injuries in the posterior hypothalamus or insomnia due to lesions of the preoptic area. The neuronal circuitries
Obesity is a new major health problem in developed countries. About 60% of American adults are obese or overweight. Obesity results from an imbalance between energy intake and energy expenditure. Excess intake over expenditure causes storage of energy into fat. While fat rich diet is one of the reasons for the increase in obesity, increasing number of studies show that the genetic background and brain function are important factors.
Most early work aiming at understanding how brain is involved
The search for new neuropeptides has taken a drastic turn in the mid of the 1990s when orphan GPCRs began to be used as targets. Since then, 13 novel neuropeptide families have been identified using the orphan receptor strategy (Table 1). Each one of these had been shown to regulate particular often several physiological responses. Interestingly, over this short period of time, several of the new neuropeptides have taken a front row in helping us understand some unresolved basic responses.
We thank our colleagues for their support during the writing of this manuscript. This work was supported by NIH Grants (MH60231, DK63001, DK70619) and a UC Discovery Grant (bio05-10485).
