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Elucidating Neurotensin Receptor cDNAs and Their Distribution

Receptors for neurotensin were identified independently in three laboratories in work published within two years of peptide sequence publication. 1-3 The localization of these receptors, their interactions with dopaminergic systems, cloning a receptor cDNA and characterizations of the distribution of its mRNA generally parallel and follow work on neurotensin peptides and their gene (TABLE 1). This chapter discusses general approaches to cloning neurotransmitter receptors, and the ways in which use of these approaches in several laboratories led to elucidation of a neurotensin receptor cDNA by expression cloning. Strategies for receptor cloning that have been used successfully include those primarily involving (a) protein purification, (b) sequence homology, (c) ligand screening, and (d) oocyte expression. Receptor purification provides the most straightforward approach to this problem. Pure protein, or enzymatic fragments of such protein, can be subjected to amino acid sequencing. With peptide or protein sequence data in hand, oligonucleotides with sequences predicted based on the protein sequence can be synthesized, radiolabeled, and allowed to hybridize with many cDNAs in libraries to identify which cDNAs might be candidate receptor clones. DNA sequences obtained from these clones can then be compared to those of other receptors and to other peptide sequences obtained during sequencing but not used to generate the screening oligonucleotides. In the case of the neurotensin receptor, as with several other receptors, protein purification approaches have proven to be time-consuming and laborious. Nevertheless, these studies have identified two protein preparations that yield binding of iodinated neurotensin with nanomolar or greater affinity. Kitabgi, Vincent, and coworkers have used affinity chromatography and other biochemical means to purify one such protein to apparent homogeneity.4 Their material displays a single binding site for radiolabeled neurotensin with a subnanomolar affinity and a molecular mass of approximately 100 kDa. Purifications performed by Mills et al. reveal a protein that displays a single site of nanomolar affinity for neurotensin, and a molecular mass in the range of 50,000 Da.5 Both of these proteins are present as single bands on SDS/polyacrylamide gel electrophoresis; protein sequence data from neither purification is currently available (but see Vincent, this volume). An alternative strategy