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Biomedical subjects

Arlene D Albert

Publications and source records attributed to Arlene D Albert.

7 recordsLinked to original sources
PubMed2006

G-protein coupled receptor structure.

Because of their central role in regulation of cellular function, structure/function relationships for G-protein coupled receptors (GPCR) are of vital importance, yet only recently have sufficient data been obtained to begin mapping those relationships. GPCRs regulate a wide range of cellular processes, including the senses of taste, smell, and vision, and control a myriad of intracellular signaling systems in response to external stimuli. Many diseases are linked to GPCRs. A critical need exists for structural information to inform studies on mechanism of receptor action and regulation. X-ray crystal structures of only one GPCR, in an inactive state, have been obtained to date. However considerable structural information for a variety of GPCRs has been obtained using non-crystallographic approaches. This review begins with a review of the very earliest GPCR structural information, mostly derived from rhodopsin. Because of the difficulty in crystallizing GPCRs for X-ray crystallography, the extensive published work utilizing alternative approaches to GPCR structure is reviewed, including determination of three-dimensional structure from sparse constraints. The available X-ray crystallographic analyses on bovine rhodopsin are reviewed as the only available high-resolution structures for any GPCR. Structural information available on ligand binding to several receptors is included. The limited information on excited states of receptors is also reviewed. It is concluded that while considerable basic structural information has been obtained, more data are needed to describe the molecular mechanism of activation of a GPCR.

Crystallography, X-Ray↗
PubMed2005

The role of cholesterol in rod outer segment membranes.

The photoreceptor rod outer segment (ROS) provides a unique system in which to investigate the role of cholesterol, an essential membrane constituent of most animal cells. The ROS is responsible for the initial events of vision at low light levels. It consists of a stack of disk membranes surrounded by the plasma membrane. Light capture occurs in the outer segment disk membranes that contain the photopigment, rhodopsin. These membranes originate from evaginations of the plasma membrane at the base of the outer segment. The new disks separate from the plasma membrane and progressively move up the length of the ROS over the course of several days. Thus the role of cholesterol can be evaluated in two distinct membranes. Furthermore, because the disk membranes vary in age it can also be investigated in a membrane as a function of the membrane age. The plasma membrane is enriched in cholesterol and in saturated fatty acids species relative to the disk membrane. The newly formed disk membranes have 6-fold more cholesterol than disks at the apical tip of the ROS. The partitioning of cholesterol out of disk membranes as they age and are apically displaced is consistent with the high PE content of disk membranes relative to the plasma membrane. The cholesterol composition of membranes has profound consequences on the major protein, rhodopsin. Biophysical studies in both model membranes and in native membranes have demonstrated that cholesterol can modulate the activity of rhodopsin by altering the membrane hydrocarbon environment. These studies suggest that mature disk membranes initiate the visual signal cascade more effectively than the newly synthesized, high cholesterol basal disks. Although rhodopsin is also the major protein of the plasma membrane, the high membrane cholesterol content inhibits rhodopsin participation in the visual transduction cascade. In addition to its effect on the hydrocarbon region, cholesterol may interact directly with rhodopsin. While high cholesterol inhibits rhodopsin activation, it also stabilizes the protein to denaturation. Therefore the disk membrane must perform a balancing act providing sufficient cholesterol to confer stability but without making the membrane too restrictive to receptor activation. Within a given disk membrane, it is likely that cholesterol exhibits an asymmetric distribution between the inner and outer bilayer leaflets. Furthermore, there is some evidence of cholesterol microdomains in the disk membranes. The availability of the disk protein, rom-1 may be sensitive to membrane cholesterol. The effects exerted by cholesterol on rhodopsin function have far-reaching implications for the study of G-protein coupled receptors as a whole. These studies show that the function of a membrane receptor can be modulated by modification of the lipid bilayer, particularly cholesterol. This provides a powerful means of fine-tuning the activity of a membrane protein without resorting to turnover of the protein or protein modification.

Animals↗
PubMed2003

A conformational trigger for activation of a G protein by a G protein-coupled receptor.

G protein-coupled receptors (GPCRs) are a family of seven transmembrane helical proteins that initiate a cellular response to an environmental signal. Once activated by an extracellular signal, GPCRs trigger the intracellular signal transduction cascade by activating a heterotrimeric G protein. The interaction between the G protein and the receptor, which triggers the signal transduction, is the focus of intense interest. Three-dimensional structures of the ground state of only one GPCR, rhodopsin, are currently available, but since the G protein cannot bind to this structure, these structures did not lead to an understanding of the activation process. The recent publication of an excited state structure for the same GPCR (and comparison to the ground state structures), in conjunction with other recent biochemical data, provides new insight into G protein activation. We find that the structure data and the biochemical data, for the first time, point to a specific mode of interaction between the G protein and the receptor. Furthermore, we find that transducin (G(t)) must alter its conformation to bind to the activated receptor; the "lock and key" fit heretofore expected is likely not the correct model. We suggest that a conformational distortion, driven by the energy of binding, is induced in G(t) when it binds to the activated receptor. The conformational change in turn enables the exchange of GTP for GDP and the dissociation of the subunits. This is an example of "induced fit" originally proposed by Koshland to describe enzyme-substrate interactions.

Animals↗
PubMed2003

A soluble peripherin/Rds C-terminal polypeptide promotes membrane fusion and changes conformation upon membrane association.

Photoreceptor rod cells contain a unique tetraspanin fusion protein known as peripherin/rds. This protein is important in membrane fusion events hypothesized to be essential to disk membrane morphogenesis and disk shedding. In vivo and in vitro fusogenic activity has been mapped to the C-terminal domain of peripherin/rds. Moreover, a fusion peptide domain localized to a 15 amino acid long region (residues 311-325) is essential for mediating lipid bilayer fusion of model membranes. To address the functional and structural properties required for peripherin/rds dependent membrane fusion, constructs of the entire C-terminal domain (residues 284-346) were generated and polypeptides expressed. A wild type-peripherin/rds C-terminal GST fusion construct that included the entire C-terminus (PERCTER) or a C-terminal truncation mutant (PERCTN) were engineered with a thrombin cleavage site. Protein expression was induced in E. coli with IPTG, expressed proteins cleaved from the GST with thrombin and purified to homogeneity on a Superdex 75 column. Purity was confirmed by SDS-PAGE and Western blot analysis. The purified wt C-terminal protein resolved as a monomer under reducing conditions on SDS-PAGE (15%) and was immunoreactive with anti peripherin/rds antibody 2B6 (gift from Dr R. Molday). The purified polypeptide promoted the requisite steps of fusion, membrane destabilization, lipid mixing and aqueous contents mixing. Conversely, the truncation mutant lacking a portion of the fusion domain was unable to promote these steps. A common feature of most membrane fusion proteins is a change in conformation upon membrane association. Structural changes in the C-terminal polypeptide were investigated using far UV CD. The far UV CD spectra of the purified C-terminal polypeptide indicated substantial alpha-helical content in the wt peptide in isotonic aqueous buffer. An increase in intensity of 208 and 222 nm CD bands upon addition of DPC vesicles indicated an increase in alpha-helical content of the polypeptide. These results demonstrate that a purified soluble form of the C-terminus of peripherin/rds can interact with biological phospholipids; moreover, this interaction promotes a conformational change that is most consistent with an increase in alpha-helical content.

Blotting, Western↗
PubMed2002

Structural studies on rhodopsin.

Bovine rhodopsin is the prototypical G protein coupled receptor (GPCR). It was the first GPCR to be obtained in quantity and studied in detail. It is also the first GPCR for which detailed three dimensional structural information has been obtained. Reviewed here are the experiments leading up to the high resolution structure determination of rhodopsin and the most recent structural information on the activation and stability of this integral membrane protein.

Amino Acid Sequence↗
PubMed2002

Structural studies of metarhodopsin II, the activated form of the G-protein coupled receptor, rhodopsin.

The structural changes that accompany activation of a G-protein coupled receptor (GPCR) are not well understood. To better understand the activation of rhodopsin, the GPCR responsible for visual transduction, we report studies on the three-dimensional structure for the activated state of this receptor, metarhodopsin II. Differences between the three-dimensional structure of ground state rhodopsin and metarhodopsin II, particularly in the cytoplasmic face of the receptor, suggest how the receptor is activated to couple with transducin. In particular, activation opens a groove on the surface of the receptor that could bind the N-terminal helix of the G protein, transducin alpha.

Amino Acid Sequence↗