Strategies for the Analysis of the Structure and Function of Cell Surface Receptors

Abstract

Intercellular communication in multicellular organisms is accomplished through the interaction of secreted hormones and growth factors with specific receptors located on the surface of target cells. These receptors are complex integral membrane glycoproteins present in extremely low abundance in the target cell plasma membrane and require special strategies for isolation as well as analysis of their structure and function. The arrangement of these proteins in the plasma membrane is determined by the topological distribution of their hydrophobic and hydrophilic domains. The structural organization of these receptors can, therefore, be inferred from an analysis of the hydrophobicity of the amino acid residues once the primary structure is determined. The elucidation of the amino acid sequence of these cell surface receptors, however, proved to be a formidable task owing to their very low abundance (less than 1 picomole/mg of purified plasma membrane protein) and their enormous size and complexity. The receptors for insulin and insulin-like growth factor 1 (IGF-1) are both heterotetrameric structures composed of two alpha-subunits of apparent molecular weight 120,000 and two beta-subunits of apparent molecular weight 90,000, held together by disulfide bridges (1–5). The determination of the amino acid sequence of these molecules could not be undertaken until the advent of recombinant DNA techniques. During the past three years we and others have succeeded in elucidating the primary structures of several cell surface receptors present only in trace quantities, by using recombinant DNA methods. The strategies are based on the isolation of cDNA clones encoding the receptor precursor by hybridization screening of a suitable cDNA library with synthetic oligonucleotide probes designed from partial amino acid sequence of the purified receptor. The elucidation of sufficiently long amino-terminal amino acid sequences as well as internal sequences by microsequencing procedures using the intact polypeptide and fragments derived from the purified receptor has been crucial to the success of this approach. The strategies that led to the successful isolation of cDNA clones for the human insulin receptor (6), the human IGF-1 receptor (7), and turkey erythrocyte ß-adrenergic receptor (8) are discussed in this article.