Question

There are two ways to study receptor responsiveness in cultured cell lines: use a cell line that naturally expresses the receptor of interest; and transfect a cell line with a gene for the receptor you are interested in in order to ensure large numbers of this receptor. Taking into consideration what you know about G protein-coupled receptors and about measurement in pharmacology, briefly discuss what the possible advantage(s) and disadvantage(s) might be of each of these approaches.

Answer 1

ANswer)

One of the greatest technical and theoretical challenges to elucidating and exploiting structure-function relationships in these systems is the emerging concept of GPCR conformational flexibility and its cause-effect relationship for receptor-receptor and receptor-effector interactions. Such conformational changes can be subtle and triggered by relatively small binding energy effects, leading to full or partial efficacy in the activation or inactivation of the receptor system at large.

GPCR structure-function. A, principal hallmarks of GPCR structure include a serpentine transmembrane topology compsed of seven ?-helical hydrophobic stretches interspersed by intra-and extracellular loops of indeterminate structure. This results in the presentation of an extracellular amino and cytoplasmic carboxyl terminus. The N terminus and extracellular loops can be glycosylated to varying degrees, and extracellular loops 1 and 2 are connected via a disulfide linkage, required for stability and function of the receptor. The C-terminal tail is anchored to the intracellular face of the lipid bilayer via palmitoylation to produce a short intracellular loop, which typically forms an ?-helical structure. These receptors also display a series of conserved amino acid motifs thought to be involved in a rearrangement of receptor domains during ligand activated signal transduction. These include three conserved motifs that provide prominent micro-switches: 1) H-III's D(E)RY motif; Arg135 when unlocked from Glu247interacts with H-V's Tyr223 to release constraints holding the receptor in the inactive state. 2) H-VI's CWxP motif; Trp265 undergoes rotamer isomerization needed for receptor activation. 3) H-VII's NPxxYx(5,6)F motif; Tyr306undergoes rotamer isomerization during activation and Asn302participates in an intrahelical H-bond network. Pro residues within H-V (Pro215), H-VI (Pro267), and H-VII (Pro303) are highly conserved and believed to form a staircase of transmembrane kinks required for the increase in rotational dynamics of these helices during activation. Rotation of these transmembrane helices brings disparate segments of intracellular loops in and out of proximity to the G protein complex causing its GTP-driven dissociation and subsequent regulation of various cellular effector cascades, the overall effect being an amplification of the original activating signal. B, the size and complexity of GPCR pharmacophores varies greatly, ranging upward from free atoms/ions to small molecules, small peptides, unmasked intrareceptor amino acid stretches, and even large glycoprotein hormones. Some correlation between receptor sequence and ligand class can be generated, which may reflect ligand-receptor recognition, but the range of sizes and diversity of ligand structures make it difficult to determine molecular mechanisms underlying ligand-receptor activation.

Pharmacological Reviews

American Society for Pharmacology and Experimental Therapeutics

The Significance of G Protein-Coupled Receptor Crystallography for Drug Discovery

Crucial as molecular sensors for many vital physiological processes, seven-transmembrane domain G protein-coupled receptors (GPCRs) comprise the largest family of proteins targeted by drug discovery. Together with structures of the prototypical GPCR rhodopsin, solved structures of other liganded GPCRs promise to provide insights into the structural basis of the superfamily's biochemical functions and assist in the development of new therapeutic modalities and drugs. One of the greatest technical and theoretical challenges to elucidating and exploiting structure-function relationships in these systems is the emerging concept of GPCR conformational flexibility and its cause-effect relationship for receptor-receptor and receptor-effector interactions. Such conformational changes can be subtle and triggered by relatively small binding energy effects, leading to full or partial efficacy in the activation or inactivation of the receptor system at large. Pharmacological dogma generally dictates that these changes manifest themselves through kinetic modulation of the receptor's G protein partners. Atomic resolution information derived from increasingly available receptor structures provides an entrée to the understanding of these events and practically applying it to drug design. Supported by structure-activity relationship information arising from empirical screening, a unified structural model of GPCR activation/inactivation promises to both accelerate drug discovery in this field and improve our fundamental understanding of structure-based drug design in general. This review discusses fundamental problems that persist in drug design and GPCR structural determination.

II. Current Challenges and Opportunities in G Protein-Coupled Receptor Drug Discovery

A. Overview of the Current G Protein-Coupled Receptor are:

1. Therapeutic Relevance.

The medicinal importance of GPCRs can be partially appreciated by considering their location and function within the cell. The physical location and disposition of GPCRs spanning the cell's plasma membrane connect extra- and intracellular environments, providing a direct mechanism for the transduction of extracellular messages into intracellular responses. In this way and together with their transmitters and effectors, GPCR systems function to modulate a broad spectrum of cellular phenomena dictated by the needs of the tissues and organs they serve. Common biological actions attributed to GPCRs include but are not limited to the following: modulation of neuronal firing, regulation of ion transport across the plasma membrane and within intracellular organelles, modulation of homeostasis, control of cell division/proliferation, and modification of cell morphology. When any of these fundamental processes go awry, the results can lead to acute or chronic human disease, a partial listing of which includes cardiovascular diseases.