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Abstract. Receptor desensitization is a key process for the protection of the cell from continuous or repeated exposure to high concentrations of an agonist. Wellestablished mechanisms for desensitization of guanine nucleotide-binding protein (G protein)-coupled receptors include phosphorylation, sequestration/internalization, and down-regulation. In this work, we have examined some mechanisms for desensitization of the cholecystokinin (CCK) receptor which is native to the pancreatic acinar cell, and have found the predominant mechanism to be distinct from these recognized processes. Upon fluorescent agonist occupancy of the native receptor, it becomes "insulated" from the effects of acid washing and becomes immobilized on the surface of the plasma membrane in a time-and temperaturedependent manner. This localization was assessed by ultrastructural studies using a colloidal gold conjugate of CCK, and lateral mobility of the receptor was assessed using fluorescence recovery after photobleaching. Of note, recent application of the same morphologic techniques to a CCK receptor-bearing Chinese hamster ovary cell line demonstrated prominent internalization via the clathrin-dependent endocytic pathway, as well as entry into caveolae (Roettger, B. F., R. U. Rentsch, D. Pinon, E. Holicky, E. Hadac, J. M. Larkin, and L. J. . J. Cell Biol. 128: 1029-1041. These organelles are not observed to represent prominent compartments for the same receptor to traverse in the acinar cell, although fluorescent insulin is clearly internalized in these cells via receptor-mediated endocytosis. In this work, the rate of lateral mobility of the CCK receptor is observed to be similar in both cell types (1-3 x 10 -1° cm2/s), while the fate of the agonistoccupied receptor is quite distinct in each cell. This supports the unique nature of desensitization processes which occur in a cell-specific manner. A plasmalemmal site of insulation of this important receptor on the pancreatic acinar cell could be particularly effective to protect the cell from processes which might initiate pancreatitis, while providing for the rapid resensitization of this receptor to ensure appropriate pancreatic secretion to aid in nutrient assimilation for the organism.G JANINE nucleotide-binding protein (G protein) 1-coupled receptors represent the largest family of receptors recognized today. They reside within the plasmalemma in a conformation which incorporates seven transmembrane helices. Agonists approach their binding sites on such molecules from the extracellular aqueous milieu, and induce a presumed conformational change of the receptor which facilitates its association with G proteins on the cytosolic face of the plasmalemma. This ternary complex of agonist-receptor-G protein typically represents the high affinity state of the receptor and a critical step in stimulus-activity coupling. Thus, agonist access

Total internal reflection fluorescence (TIRF) microscopy is used to measure the dissociation kinetic rate of fluorescein-labeled epidermal growth factor from its specific receptors on the surface of intact but mildly fixed A431 human epidermoid cells in culture. Prior applications of TIRF microscopy have been limited to nonreceptor binding or to model membrane systems. The evanescent field excites fluorescence selectively at the surface of the cell proximal to the coverslip. "Prismless" epiillumination TIR is employed to avoid space limitations and is achieved by passing the excitation laser beam through a high (1.4)-aperture objective so that the light is incident at the glass/water interface beyond the critical angle. Long-term focus is maintained by a special feedback system. Of the possible effects that can influence the time course of the postbleach fluorescence recoveries-the EGF/receptor dissociation ratek 2, the bulk solution diffusion rate of EGF, and the cell surface motion of the receptors-we infer that the dissociation ratek 2 dominates. Several fitting schemes are compared and indicate the presence of a multiplicity of values fork 2, ranging from about 0.05 to 0.004 s(-1), with an average value of about 0.012 s(-1). These results compare well with values previously obtained by radiolabel/washing techniques. The significance of the results in terms of kinetic models and the advantages of the TIRF technique for these sorts of measurements are discussed.

We investigate the dynamics of a synthetic genetic repressilator with quorum sensing feedback. In a basic genetic ring oscillator network in which three genes inhibit each other in unidirectional manner, an additional quorum sensing feedback loop stimulates the activity of a chosen gene providing competition between inhibitory and stimulatory activities localized in that gene. Numerical simulations show several interesting dynamics, multi-stability of limit cycle with stable steady-state, multi-stability of different stable steady-states, limit cycle with period-doubling and reverse period-doubling, and infinite period bifurcation transitions for both increasing and decreasing strength of quorum sensing feedback. We design an electronic analog of the repressilator with quorum sensing feedback and reproduce, in experiment, the numerically predicted dynamical features of the system. Noise amplification near infinite period bifurcation is also observed. An important feature of the electronic design is the accessibility and control of the important system parameters.