Our
laboratory
is broadly interested in understanding the biochemistry, subcellular
organization and dynamics of receptor-mediated signaling systems in
eukaryotic cells. Cell signaling in complex cells typically exhibits
precise spatiotemporal control, and such organization is essential to
acute signaling. Dynamic reorganization of the cellular signaling
machinery occurs throughout life, and contributes to physiological
homeostasis and plasticity occurring over a wide range of time scales.
In addition, these processes can be profoundly altered under
pathological conditions, and influenced in interesting ways by acute or
chronic administration of clinically relevant drugs. Much
of our lab's work has focused on the subcellular organization and
membrane trafficking of seven-transmembrane signaling receptors (7TMRs),
traditionally called G protein-coupled receptors (GPCRs). 7TMRs
comprise the largest family of signaling receptors, and individual
members of this family function in the control of virtually all aspects
of animal physiology. 7TMRs are also molecular targets of a wide range
of therapeutic and illicit drugs. We are also interested in other types
of signaling receptor, such as glutamate-regulated ion channels that
function at neuronal synapses, and membrane proteins such as connexins
that function in direct cell-cell communication. Our
studies have contributed to understanding how particular signaling
receptors, and other cellular proteins with which they interact, are
organized
and functionally regulated in individual cells. In essence they reveal
an intricate choreography of regulated membrane trafficking processes,
which contribute to dynamic reorganization of the cell's signaling
machinery under both physiological and pathological conditions. In many
cases this reorganization by membrane traffic occurs in a highly
receptor-specific manner, and we are intrigued that - in some cases-
dynamic reorganization of the same signaling system can occur
differentially in response to physiological compared to pharmacological
stimulation. We believe that such processes of regulated membrane trafficking, besides contributing at a fundamental level to diverse aspects of physiological homeostasis and plasticity, are important to elucidating pathological processes and the cellular basis of drug action. It has emerged relatively recently that various signaling receptors, while they are delivered and reorganized largely via the cell's 'core' membrane trafficking machinery, can produce 'customized' effects by exerting local control over the function or dynamics of this machinery. Thus we think that understanding the regulated trafficking of particular signaling receptors can inform us, more generally, about the relationship between conserved membrane trafficking mechanisms and their relationship to the diversity of 'cargo' that they transport. Main scientific goals of our present work include (1) defining individual steps of receptor membrane trafficking, (2) determining points of regulation by physiological and pharmacological stimuli, and (3) elucidating 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. We are particularly interested in understanding functional 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 can be quite
challenging to unambiguously link specific cellular regulatory
mechanisms elucidated in individual cells to salient physiological and
behavioral effects occurring in neural circuits and in vivo. This
challenge gets even more interesting when one begins to consider
pharmacological and pathological effects. Understanding the physiological consequences of membrane
traffic to the diverse signaling activities of seven-transmembrane
receptors has continued to pose fascinating challenges. As a first step,
we have focused on defining precisely whether, and how, particular
regulatory mechanisms defined in cell culture models (or in cell-free
systems) occur in the
context of native cell types and tissues. These studies have verified
the occurrence
of regulated endocytic trafficking of a number of receptors in vivo,
yielded further insight regarding differences among the regulatory
effects of
certain 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 and signaling with improved
spatiotemporal resolution, and in differentiated membrane
domains. Efforts in this direction have stimulated, and been greatly
aided by, terrific collaborations with colleagues at UCSF and elsewhere. One recent direction in the lab is the application of chemical genetic approaches as a means to extend pharmacology and to precisely manipulate defined receptor regulatory mechanisms. We are working toward application of these approaches to ex vivo tissue preparations as well as to intact animals. Goals of this fledgling effort are to further move mechanistic cell biology toward the in vivo realm, and to develop improved tools for assessing the potential of specific receptor regulatory mechanisms as future therapeutic targets. |