The process of synthesis, insertion into the plasma membrane, and degradation of beta-adrenergic receptors will be studied in cultured human astrocytoma cells (line 1321N1). Previous studies have demonstrated that beta-adrenergic receptors are expressed differentially during growth of 1321N1 cells in culture. Using these growth-related changes in receptor turnover as a model system, we will investigate the kinetics of receptor accumulation in relation to synthesis, plasma membrane insertion and degradation of the receptor. The metabolic fate of newly synthesized receptors will be examined directly using stable isotope labelled amino acids. Cells will be incubated with 2H-, 13C- and 15N-labelled amino acids, and heavy versus light receptors will be identified after separation by gradient centrifugation of detergent solubilized, 125IHYP-labelled, receptors. We will also use the irreversible beta-adrenergic receptor antagonist, NMNP-NBE, to inactivate existing receptor sites in a number of these experiments. Using these techniques, the processing of newly synthesized receptor protein will be quantitatively followed under a variety of experimental paradigms. The contribution of changes in relative rates of receptor synthesis and degradation to the marked loss of receptors that occurs during catecholamine-induced desensitization will be examined. The functional significance of any intracellular pool of receptors will be examined as it relates to receptor synthesis, insertion, recycling, and degradation. The relationship of such an intracellular pool to a membrane fraction of receptors which does not copurify with plasma membrane fractions on sucrose density gradients will be assessed. Drugs that affect translation or transcription will be utilized to investigate the role or protein and RNA synthesis in the regulation of the assembly of receptors. Synthesis, incorporation and degradation will also be examined under conditions in which pH, temperature, intracellular ATP levels, and microtubule integrity are altered.