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Many ligands, however, bind to their receptors with much lower affinity. If the Kd for binding is greater than = 1 X 10~7 M, any ligand bound to receptors is likely to dissociate in the few seconds it takes to separate the cells (e.g., by centrifugation) from free (unbound) ligand and measure the amount of bound ligand. One way to detect weak binding of a ligand to its receptor is in a competition assay with another ligand that binds to the same receptor with high affinity (low Kd value). In this type of assay, increasing amounts of an unlabeled, low-affinity ligand (the competitor) are added to a cell sample with a constant amount of the radiolabeled, high-affinity ligand (Figure 13-5). Binding of unlabeled competitor blocks binding of the radioactive ligand to the receptor; the concentration of competitor required to inhibit binding of half the radioactive ligand approximates the Kd value for binding of the competitor to the receptor.

Synthetic analogs of natural hormones are widely used in research on cell-surface receptors and as drugs. These analogs fall into two classes: agonists, which mimic the function of a natural hormone by binding to its receptor and inducing the normal response, and antagonists, which bind to the receptor but induce no response. By occupying ligand-binding sites on a receptor, an antagonist can block binding of the natural hormone (or agonist) and thus reduce the usual physiological activity of the hormone.

For instance, addition of two methyl groups to epineph-rine generates isoproterenol, an agonist that binds to epi-nephrine receptors on bronchial smooth muscle cells about tenfold more strongly than does epinephrine (see Figure 13-5). Because ligand binding to these receptors promotes relaxation of bronchial smooth muscle and thus opening of the air passages in the lungs, isoproterenol is used in treating bronchial asthma, chronic bronchitis, and emphysema. Activation of epinephrine receptors on cardiac muscle cells increases the contraction rate. The antagonist alprenolol and related compounds, referred to as beta-blockers, have a very high affinity for these epinephrine receptors. Such antagonists are used to slow heart contractions in the treatment of cardiac arrhythmias and angina. I

Receptors Can Be Purified by Affinity Techniques or Expressed from Cloned Genes

Cell-surface receptors often can be identified and followed through isolation procedures by affinity labeling. In this technique, cells are mixed with an excess of a radiolabeled ligand for the receptor of interest. After unbound ligand is washed away, the cells are treated with a chemical agent that covalently cross-links bound labeled ligand molecules and receptors on the cell surface. Once a radiolabeled ligand is co-valently cross-linked to its receptor, it remains bound even in the presence of detergents and other denaturing agents that are used to solubilize receptor proteins from the cell membrane. The labeled ligand provides a means for detecting the receptor during purification procedures.

Another technique often used in purifying cell-surface receptors that retain their ligand-binding ability when solu-bilized by detergents is similar to affinity chromatography using antibodies (see Figure 3-34). To purify a receptor by this technique, a ligand for the receptor of interest, rather than an antibody, is chemically linked to the beads used to form a column. A crude, detergent-solubilized preparation of membrane proteins is passed through the column; only the receptor binds, and other proteins are washed away. Passage of an excess of the soluble ligand through the column causes the bound receptor to be displaced from the beads and eluted from the column. In some cases, a receptor can be purified as much as 100,000-fold in a single affinity chromatographic step.

Once a receptor is purified, its properties can be studied and its gene cloned. A functional expression assay of the cloned cDNA in a mammalian cell that normally lacks the encoded receptor can provide definitive proof that the proper protein indeed has been obtained (Figure 13-6). Such expression assays also permit investigators to study the effects of mutating specific amino acids on ligand binding or on "downstream" signal transduction, thereby pinpointing the receptor amino acids responsible for interacting with the lig-and or with critical signal-transduction proteins.

The cell-surface receptors for many signaling molecules are present in such small amounts that they cannot be purified by affinity chromatography and other conventional biochemical techniques. These low-abundance receptor proteins can now be identified and cloned by various recombinant DNA techniques, eliminating the need to isolate and purify them from cell extracts. In one technique, cloned cDNAs prepared from the entire mRNA extracted from cells that produce the receptor are inserted into expression vectors by techniques described in Chapter 9. The recombinant vectors then are transfected into cells that normally do not synthesize the receptor of interest, as in Figure 13-6. Only the very few transfected cells that contain the cDNA encoding the desired receptor synthesize it; other transfected cells produce irrelevant proteins. The rare cells expressing the desired receptor can be detected and purified by various techniques such as fluorescence-activated cell sorting using a fluorescent-labeled ligand for the receptor of interest (see Figure 5-34). Once a cDNA clone encoding the receptor is identified, the sequence of the cDNA can be determined and that of the receptor protein deduced from the cDNA sequence.

Genomics studies coupled with functional expression assays are now being used to identify genes for previously unknown receptors. In this approach, stored DNA sequences are analyzed for similarities with sequences known to encode receptor proteins (Chapter 9). Any putative receptor genes that are identified in such a search then can be tested for their ability to bind a signaling molecule or induce a response in cultured cells by a functional expression assay.

Receptor for ligand other than X

No binding of X; no cellular response

Transfection with cDNA expression vector and selection of transformed cells

Transfection with cDNA expression vector and selection of transformed cells

▲ EXPERIMENTAL FIGURE 13-6 Functional expression assay can identify a cDNA encoding a cell-surface receptor.

Target cells lacking receptors for a particular ligand (X) are stably transfected with a cDNA expression vector encoding the receptor. The design of the expression vector permits selection of transformed cells from those that do not incorporate the vector into their genome (see Figure 9-29b). Providing that these cells already express all the relevant signal-transduction proteins, the transfected cells exhibit the normal cellular response to X if the cDNA in fact encodes the functional receptor.

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