Our laboratory is broadly interested in understanding the subcellular organization and dynamics of receptor-mediated signaling systems in eukaryotic cells. These processes are essential to the short-term regulation of cellular signaling, as well as to longer-term changes in receptor number and function in specific membrane domains. Our primary focus is on endocytic membrane trafficking of catecholamine and opioid receptors, two important classes of G protein-coupled signaling receptor. It turns out that these receptors are exquisitely organized in vivo, and undergo regulated membrane trafficking processes in a receptor-specific and ligand-specific manner. Some main scientific goals of our present work are to (1) define individual steps of receptor membrane trafficking, (2) determine points of regulation by physiological and pharmacological stimuli, and (3) elucidate key regulated trafficking events at the level of biochemical mechanism. Our methods include a variety of biochemical, molecular biological, immunochemical, pharmacological, and microscopic imaging techniques applied to cultured cell models.
We
are also interested in understanding physiological consequences of the
regulatory processes that we study. A rich background in receptor cell
biology suggests that endocytic trafficking of receptors plays a
fundamental role in regulating the intensity of signaling responses
elicited by specific inputs. Two main effects have been established,
and are readily understood in terms of classical receptor theory: (1) a
change in the maximal response that can be achieved by a saturating
concentration of agonist (sometimes called ''system efficacy" of a
particular signaling response) and (2) a change in the effective
potency with which a particular agonist can produce a given signaling
response. Both of these effects are thought to occur in vivo, and may
contribute to clinically important phenomena such as tachyphylaxis and
tolerance to various drugs. An evolving area of research suggests, in
addition, a distinct role of regulated trafficking in qualitatively
"switching" receptor signaling specificity from one downstream effector
pathway to another.
Despite close
relationships between mechanism and function apparent from studies of
cultured cells, and extensive effort by many groups, it has been
remarkably difficult to unambiguously link specific cellular regulatory
mechanisms to physiological and behavioral effects in vivo. This is
particularly true for regulatory events occurring in the nervous
system, where many G protein-coupled receptors affect synaptic
transmission indirectly. We are very interested in this problem and are
taking several approaches to address it.
One
approach takes advantage of the fact that, as it turns out, certain
ligand-gated ion channels that directly mediate synaptic transmission
are regulated in cultured cells by endocytic membrane trafficking.
Studies carried out in collaboration with colleagues in Roger Nicoll's
and Rob Malenka's labs have established a direct role of endocytic
membrane trafficking of one class of such receptors, AMPA-type
glutamate receptors, in activity-dependent synaptic plasticity
occurring in an acute slice preparation of rat hippocampus.
To move forward with understanding physiological consequences of these regulatory mechanisms to signaling by G protein-coupled receptors in particular, we have begun by defining precisely whether, and how, specific regulatory mechanisms studied in cell culture occur in the context of native tissues. These studies have verified the occurrence of regulated endocytic trafficking of opioid receptors in vivo, yielded new information regarding differences among the regulatory effects of certain opioid drugs, and revealed that the cell biology of receptor regulation occurring in physiologically relevant CNS neurons is even more precisely controlled than observed in heterologous cell models. These efforts have also motivated us to apply new imaging methods to examine the dynamics of receptor trafficking in differentiated membrane domains of neurons.
We are also beginning to explore a variety of chemical and structural approaches for manipulating defined receptor regulatory mechanisms, and the application of these approaches to ex vivo tissue preparations as well as intact animals. Goals of this latter effort are to move mechanistic cell biology truly into the in vivo realm, and to develop improved tools for assessing the potential of specific receptor regulatory mechanisms as future therapeutic targets.