G-Protein-Coupled Receptors: Molecular Mechanisms Involved in Receptor Activation and Selectivity of G-Protein Recognition

Title: G-Protein-Coupled Receptors: Molecular Mechanisms Involved in Receptor Activation and Selectivity of G-Protein Recognition
Author: Wess, Jurgen
Publisher: Faseb Journal
Date Published: April 01, 1997
Reference Number: 246
G-protein-coupled receptors (GPCRs) play fundamental roles in regulating the activity of virtually every body cell. Upon binding of extracellular ligands, GPCRs interact with a specific subset of heterotrimeric G-proteins that can then, in their activated forms, inhibit or activate various effector enzymes and/or ion channels. Molecular cloning studies have shown that GPCRs form one of the largest protein families found in nature, and it is estimated that approximately 1000 different such receptors exist in mammals. The molecular mechanisms involved in GPCR function, particularly the molecular modes of receptor activation and G-protein recognition and activation, have therefore become the research focus of an ever increasing number of laboratories. This review will summarize and attempt to integrate recent data derived from structural, molecular genetic, biochemical, and biophysical studies that have shed new light on these processes.
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This translation by the NDI Foundation is to assist the lay reader. To provide a clear, accessible interpretation of the original article, we eliminated or simplified some technical detail and complicated scientific language. We concentrated our translation on those aspects of the article dealing directly with NDI. The NDI Foundation thanks the researchers for their work toward understanding and more effectively treating this disorder.
© Copyright NDI Foundation 2007 (JC)

Receptors are molecular structures located within a cell or in the cell membrane (cell surface) that form a weak, reversible bond with an extracellular agent such as a hormone, neurotransmitter or antigen. Each receptor is designed to bind only with a specific agent. For example, the vasopressin-2 receptor (V2R) will only bind with the antidiuretic hormone, arginine vasopressin (AVP).

There is a family of receptors known as G-protein-coupled receptors (GPCRs). These receptors link with G-proteins in order to pass on the signal from the extracellular agent with which the receptor has formed a bond. For example, when AVP binds with V2R (which is a G-protein-coupled receptor), the V2R is activated and couples with a Gs protein. Once coupled, it can pass on the signal from AVP to the rest of the molecular sequence that effects an antidiuretic action.

There are many different classes of G-proteins within the G-protein family. For example, there are Gs-proteins, Gi-proteins and Gq/11-proteins. All G-proteins are located in the side of the cell membrane closest to the inside of the cell. (The cell membrane is the thin strip of lipid that encircles a cell to form the cell's circumference, separating it from the extracellular environment.)

The activity of every body cell is regulated by extracellular signals. The majority of these signals are transmitted into the cell interior by means of GPCRs. Researchers, discovering the importance of GPCRs, seek to understand more about them.

In his article, Wess reviews and discusses the current understanding of the molecular mechanisms involved in GPCR function, focusing primarily on what structural changes GPCRs undergo when activated, and how they recognize and activate their respective G-proteins.

Even though there are many different types of GPCRs, each of them shares a similar molecular structure. The same applies to G-proteins. All classes of G-proteins are composed of three subunits, called alpha, beta and gamma. Each GPCR is a string of amino acid residues, varying in length according to the number of residues that make it up. For example, a V2R is comprised of 371 amino acid residues. (An amino acid loses a water molecule when it joins with another amino acid. The portion of the amino acid which remains is called a residue.) A goodly portion of the string lies within the cell membrane in seven distinct coils called transmembrane domains 1 - 7. Part of the string snakes outside the cell membrane into the extracellular environment, forming three curves called extracellular loops 1 - 3. Part of it snakes within the cell, forming three curves called intracellular loops 1 - 3. One end of the GPCR, called the amino-terminus, lies outside the cell with the extracellular loops. And the other end, called the carboxy-terminus, lies inside the cell with the intracellular loops. (Please refer to V2R for an visual example of a GPCR.)

Knowing the orientation of the GPCR and what amino acid is located at each point along the GPCR's length provides an enormous amount of detail and insight into the molecular mechanics involved in the coupling of GPCRs and G-proteins.

Just as a person must cup her hands to hold water in them, so a GPCR must manipulate its shape to couple with its G-protein. This changing of position of the GPCR from its resting state to its active state generally occurs after the GPCR is activated by binding with the extracellular agent specific to it. Research indicates that the shift in shape is minor, but significant. For example when the GPCR rhodopsin is activated, a relatively small outward movement of the portion of the sixth transmembrane domain that is near the cell cytoplasm occurs. Its effect is to open its intracellular surface to the point where the G-protein is able to interact with previously inaccessible amino acids that comprise specific regions of the receptor. Receptor activation resulting in shape changes that open previously inaccessible amino acids to the G-protein is held to occur in all GPCRs. An analogous image might be a flower bud opening enough to let a bee take its pollen.

Discovering the structure of both GPCRs and G-proteins has allowed researchers to design experiments that have revealed the amino acids and the specific regions of certain GPCRs that are involved in such activities as receptor activation and GPCR/G-protein coupling. For example, in most cases, an individual activated GPCR can recognize and activate only a limited set of the many structurally closely related G-proteins. Many studies have indicated that the selectivity of G-protein recognition is primarily determined by amino acids located in a GPCR's intracellular loop 2 and certain portions of the intracellular 3 domain.

Mutational analysis has identified a series of single amino acids that are critical for GPCRs to properly recognize their G-protein. Residues at the junction of the third intracellular loop and the cell membrane, and amino acids in the second intracellular loop have been so identified. Similarly, mutational analysis has revealed some of the amino acid residues in GPCRs generally required for them to efficiently couple with and activate their G-proteins. For example, when researchers replaced an arginine amino acid residue with a different amino acid, it virtually abolishes G-protein-coupling. Thus, this arginine is seen as critical for G-protein coupling. Most GPCRs contain a stretch of charged residues in a portion of the third intracellular loop. One or more positively charged residues within this motif are critical for efficient G-protein activation.

Researchers are also looking into the portions of the G-protein and GPCR that come into contact with each other, but their work is hampered because they have not as yet identified the specific regions on the G-protein that are in contact with the various functionally critical receptor sites. Still, progress is being made. There is, for example, evidence indicating that the m2 muscarinic receptor requires a four-amino acid motif (valine, threonine, isoleucine, leucine) located at the junction of the third intracellular loop and the sixth transmembrane domain. Researchers speculate that this amino acid sequence becomes accessible to the carboxy-terminus of the alpha subunit of the Gi/o-protein upon m2 muscarinic receptor interaction. This interaction determines that the receptor will couple with this G-protein and then activate the G-protein. There is more evidence that suggests the beta and gamma subunits (they never separate from one another and are often referred to as the beta/gamma complex) also directly contact the receptor and that receptor/G-protein coupling selectivity may depend on all three subunits of the G-protein.

Further research might lead to effective therapeutic strategies of human diseases in which dysfunctions of distinct receptor/G-protein pairs are involved. For example, some mutations of the V2R gene result in V2Rs incapable of coupling with and activating its Gs protein. Because of this, AVP cannot get its signal through to the molecular sequence that leads to the kidneys being able to reabsorb body water flowing through the kidney collecting ducts. Thus, the kidneys cannot concentrate urine and their ability to maintain body water balance is impaired. This disorder is called nephrogenic diabetes insipidus (NDI). It is hoped that at some point genetic therapies will be developed to effectively treat or cure this disease and all others where receptor/G-protein interactions are involved.