Receptors were initially regarded as theoretical entities until the 1970s when labelling experiments allowed us to identify them as biological realities. Radioactive ligands made it possible to extract and purify receptor material. This procedure was used to first identify the Nicotinic Acetylcholine receptor.

Tissues containing the radiolabelled receptors were then treated with non-ionic detergent to render the membrane-bound receptor protein soluble. The receptors were then purified by the technique of affinity chromatography in which a receptor ligand, bound covalently to the matrix of a chromatography column, is used to absorb the receptor and separate it from other substances in the extract. The receptor was then eluded from the column by flushing it through with a solution containing an antagonist.

After isolating and purifying the receptors it was then possible to analyse the amino acid sequences allowing the corresponding base sequence of mRNA to be deduced (with some ambiguity, due to the differences in the genetic code). Oligonucleotide probes were then synthesised and used to extract the full length DNA sequence by cloning methods.

DNA is cloned with the aid of special enzymes called restriction enzymes. Each enzyme recognises only a specific base sequence of DNA and isolates that particular DNA fragments. The fragments generated by a restriction enzyme acting on one DNA molecule will have the same set of single stranded ends as the fragments produced by the same enzyme acting on a different DNA molecule (providing both DNA molecules have sequences recognised by the enzyme). Therefore fragments from the DNA molecules of 2 different organisms (e.g. bacterium and frog) can be joined by renaturation of complementary single stranded fragments. This joining technique can be particularly useful if one of the sources of DNA cut (or cleaved) is a plasmid (see diagram below). Plasmids are extra chromosomal circular DNA molecules found in most bacterial species (and also in some species of eukaryotes) and are useful tools for molecular biologists. Using plasmids cleaved by restriction enzymes at one particular site and mixing this linnearized plasmid DNA with frog DNA cut with the same restriction enzyme, a circular plasmid DNA molecule containing frog DNA can be generated. The importance of this technique is that the hybrid plasmid can be re-established in a bacterium and the inserted frog DNA fragment, which replicates as part of the plasmid, is cloned.

 

Diagram from Molecular Biology (2nd edition) David Freifelder

The first receptor clones were obtained in this way but now science offers us a number of alternatives. For example expression cloning involves the use of a cloning vector or host system that allows the production of the protein encoded by the cDNA. If the protein (e.g. receptor) is expressed directly, in a bacterial cell, antibodies against the receptor protein may be used to detect bacterial clones carrying the correct cDNA. Otherwise the cDNA may be transcribed artificially to produce mRNA, which is then injected into a frog oocyte. The mRNA corresponding to the receptor protein is translated by the oocyte, and the protein is expressed on its surface, where its presence can be detected by recording changes in membrane potential or conductance in response to application of the relevant agonist. This technique, though laborious, is widely used, because it dispenses with the need to purify the receptor protein and obtain antibodies to it.

It is also possible to introduce foreign DNA into mammalian cell lines by tranfection, and to monitor receptor expression by the appearance of specific ligand binding. This technique is often used to study the binding and pharmacological characteristics of the receptors that have been cloned by the methods described above.

Cloning strategies, which require neither protein purification or expression systems, have also been used successfully. These are based on expected sequence similarities or homologies between the receptor that is sought and those already identified. A region of sequence homology allows, by the use of PCR (polymerase chain reaction) and RACE (rapid amplification of cDNA ends), replication of DNA molecules that contain that sequence. If the chosen sequence is, for example, one that is conserved in several adrenoceptors, what is amplified may well turn out to be another new adrenoceptor or it may turn out to be something quite different. Unexpected receptors (e.g. cannabinoid receptor) are sometimes found by accident in this way, and there are several examples of receptor like structures for which no functional ligand is known.

The cloning of receptors has often brought to light molecular variants (subtypes) of known receptors, which have not been identified from pharmacological studies. This tends to produce some classification confusion, however the molecular characteristics of receptors are essential in the long term. Molecular biologists remain undaunted by the explosion of receptor subtypes where as pharmacologists remain sceptical about the clinical applications of such findings.