Receptor heterodimerization: a new level of cross-talk
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《临床调查学报》
National Heart and Lung Institute, Imperial College, London, United Kingdom.
Abstract
Most G protein–coupled receptors (GPCRs) probably exist as homodimers, but it is increasingly recognized that GPCRs may also dimerize with other types of GPCRs and that this physical interaction may affect the function of either receptor. A study in this issue of the JCI demonstrates how heterodimerization between prostaglandin E receptors and 2–adrenergic receptors (2ARs) in airway smooth muscle cells results in uncoupling of 2ARs and a diminished bronchodilator response to 2AR agonists. This illustrates what we believe to be a novel mechanism of receptor cross-talk and highlights the potential importance of GPCR heterodimerization in diseases such as asthma and how this could lead to the development of more specific therapies in the future.
See the related article beginning on page 1400
Cross-talk between different receptors has long been recognized as an important determinant of cellular response in health and disease. Traditionally this cross-talk has been explained by interaction of intracellular signal transduction pathways, phosphorylation of receptors and regulatory proteins by kinases, or effects on intracellular calcium release (1). Receptor cross-talk represents a means of fine-tuning the control of cellular function and is relevant to understanding disease and response to therapeutic agents that interact with cell-surface receptors. Recently there has been growing recognition that physical interaction between cell surface receptors may be a novel means of receptor cross-talk, and this has been studied in the greatest detail for G protein–coupled receptors (GPCRs).
Approximately 400 GPCRs are known to mediate the effects of endogenous ligands and are the targets for about half of currently used prescription drugs (2-4). The interaction of an agonist with the binding pocket of a GPCR induces a conformational change in the transmembrane-spanning segments. This results in its association with a G protein that leads to activation of a signal transduction pathway, resulting in the characteristic cellular response. GPCRs were conventionally thought to exist and act as monomers, but there is accumulating evidence that most GPCRs probably exist as dimers or even oligomers (5-7). Furthermore, different GPCRs may interact with each other, forming heterodimers. This has important implications for understanding cellular regulation and the action of agonists. Dimerization of GPCRs was first proposed by Agnati and colleagues in the 1980s (8), based on the finding of unexplained cooperativity between certain agonists and a larger-than-expected molecular size of receptor proteins observed by gel electrophoresis. However, this idea received little attention until the last decade.
Receptor homodimerization
Although the idea of receptor dimerization was at first resisted and then thought to be the exception or a feature of artificial overexpression systems, it is now clear that most GPCRs exist as homodimers for at least some period during their existence. This has most convincingly been demonstrated using fluorescence resonance energy transfer or bioluminescence resonance energy transfer (BRET), which directly demonstrate receptor protein dimerization in living cells. Indeed, studies using BRET indicate that more than 80% of 2–adrenergic receptors (2ARs) exist as homodimers (9). This dimerization appears to occur during the synthesis of receptors in the endoplasmic reticulum and is necessary for the transport of the newly formed receptors to the cell surface (5-7). Furthermore, interfering with receptor homodimerization affects the trafficking and function of the receptor (7). The sites of physical interaction between GPCRs appear to be the transmembrane -helices. Using a peptide that corresponds to transmembrane helix VI of the 2AR in order to interfere with the presumed dimerization sequence, there was a significant reduction in the incidence of dimerization, and this was associated with reduced activation of adenylyl cyclase by 2AR agonists (10). This suggests that high-affinity binding of receptor and G protein may require the GPCR dimer. Furthermore, mutation of the putative dimerization motif in helix VI prevented the movement of 2ARs from their site of synthesis in the endoplasmic reticulum to the cell surface, indicating that homodimerization is critical for cell-surface expression of 2ARs (11). There is debate about the effect of agonists on the extent of receptor dimerization, but most studies indicate that dimers are formed during receptor synthesis in the endoplasmic reticulum and are formed in the absence of agonist stimulation (7).
Receptor heterodimerization
There is increasing evidence that different GPCRs may form heterodimers and that this can affect the function of each agonist, resulting in significant functional interactions. Indeed, this phenomenon may account for some drug interactions that were unexpected or previously difficult to explain. Many different GPCR heterodimers have now been described (5-7), but the functional consequence of heterodimerization is not predictable. 2ARs may interact with both - and -opioid receptors. This does not appear to affect the binding or effects of agonists but has an effect on receptor trafficking (12). When 2ARs are coexpressed with -opioid receptors, both a 2AR-agonist and a -opioid agonist cause downregulation and internalization of the -opioid receptor, whereas when coexpressed with -opioid receptors, neither agonist causes receptor internalization (12). The 1D–adrenergic receptor is not normally expressed on the cell surface of most cells unless it dimerizes with 1B receptors or 2ARs, indicating an important role for heterodimerization in the trafficking of certain receptors from the endoplasmic reticulum to the cell surface (13, 14). A particular GPCR can only dimerize with certain other GPCRs; receptors that are closely related in structure in general have a higher affinity for interaction, so that homodimers form more readily than heterodimers. However, 2ARs may heterodimerize with 1ARs and 3ARs with a similar affinity to themselves, which suggests that at equal levels of expression, an equal number of homodimers and heterodimers is likely to exist (9, 15). There is still little known about how heterodimerization affects receptor function and signaling. Heterodimerization between 2ARs and angiotensin II type 1 (AT1) receptors appears to account for the crossover effects of beta blockers and AT1 receptor blockers on myocardial function. A beta blocker reduces the effect of angiotensin II in murine cardiomyocytes in vitro and on cardiac function in vivo, whereas an AT1 receptor blocker inhibits the response to a -agonist (16).
Receptor heterodimerization in airway smooth muscle
Airway smooth muscle tone is regulated by multiple GPCRs, and cross-talk between different classes of receptor, such as 2ARs and muscarinic M3 receptors, has previously been demonstrated (17). These interactions may be of clinical relevance in asthma, where airway smooth muscle tone is increased. In this issue of the JCI, McGraw and colleagues demonstrate a novel type of cross-talk between GPCRs involving receptor heterodimerization and demonstrate its functional consequences on murine airway smooth muscle contraction (18). PGE2 activated the PGE2 receptor, EP1 subtype (EP1R) on airway smooth muscle cells, which is coupled to Gq and calcium ion release and yet did not elicit contraction as expected. However, PGE2 reduced the bronchodilator response to a 2AR agonist by attenuating the increase in cAMP. Forskolin, a direct activator of adenylyl cyclase, was unaffected, indicating an upstream interaction between PGE2 and 2AR agonist. Using fluorescence microscopy in airway smooth muscle cells, and BRET and coimmunoprecipitation in a cell line, heterodimerization between EP1Rs and 2ARs was demonstrated. EP1R agonists bind to the heterodimer and uncouple 2ARs from Gs, thus diminishing the bronchodilator response of the 2AR agonist (Figure 1). This represents a novel level of functional antagonism between bronchoconstrictor and bronchodilator mechanisms and may contribute to the reduced response to 2AR agonists that may occur in severe asthma, when endogenous concentrations of PGE2 may be elevated. Since airway smooth muscle cells express more than 30 different GPCRs, many other possible GPCR heterodimerizations remain to be explored (19).
Clinical implications
At present there is relatively little information about the functional consequences of receptor heterodimerization, but the study by McGraw et al. (18) demonstrates that such receptor interactions may have important functional consequences. Receptor heterodimerization may affect the surface expression of receptors, the rate of receptor desensitization, and the effect of agonists on signal transduction, resulting in several different and so-far unpredictable functional consequences. This allows for the possibility of finding unexpected drug interactions or novel therapeutic agents that selectively activate certain heterodimer pairs. Since the relative expression of different GPCRs in various cell types differs, this makes it potentially possible to develop more selective drugs in the future. The role of receptor heterodimerization in disease has hardly been explored, but genetic polymorphisms in areas of the receptor that affect dimerization with other receptors may alter the function of the receptor, as has already been demonstrated for chemokine receptors (6, 20). GPCR heterodimerization appears to be a novel means of cell regulation that is likely to have clinical and therapeutic significance in the future.
Footnotes
Nonstandard abbreviations used: 2AR, 2–adrenergic receptor; AT1, angiotensin II type 1; BRET, bioluminescence resonant energy transfer; EP1R, PGE2 receptor, EP1 subtype; GPCR, G protein–coupled receptor.
Conflict of interest: The author has declared that no conflict of interest exists.
References
Vazquez-Prado, J., Casas-Gonzalez, P., and Garcia-Sainz, J.A. 2003. . G protein-coupled receptor cross-talk: pivotal roles of protein phosphorylation and protein-protein interactions. Cell. Signal. 15::549-557.
Drews, J. 1996. . Genomic sciences and the medicine of tomorrow. Nat. Biotechnol. 14::1516-1518.
Pierce, K.L., Premont, R.T., and Lefkowitz, R.J. 2002. . Seven-transmembrane receptors. Nat. Rev. Mol. Cell Biol. 3::639-650.
Hill, S.J. 2006. . G-protein-coupled receptors: past, present and future. Br. J. Pharmacol. 147(Suppl. 1)::S27-S37.
Milligan, G. 2004. . G protein-coupled receptor dimerization: function and ligand pharmacology. Mol. Pharmacol. 66::1-7.
Prinster, S.C., Hague, C., and Hall, R.A. 2005. . Heterodimerization of G protein-coupled receptors: specificity and functional significance. Pharmacol. Rev. 57::289-298.
Bulenger, S., Marullo, S., and Bouvier, M. 2005. . Emerging role of homo- and heterodimerization in G-protein-coupled receptor biosynthesis and maturation. Trends Pharmacol. Sci. 26::131-137.
Agnati, L.F., Fuxe, K., Zoli, M., Rondanini, C., and Ogren, S.O. 1982. . New vistas on synaptic plasticity: the receptor mosaic hypothesis of the engram. Med. Biol. 60::183-190.
Mercier, J.F., Salahpour, A., Angers, S., Breit, A., and Bouvier, M. 2002. . Quantitative assessment of 1- and 2-adrenergic receptor homo- and heterodimerization by bioluminescence resonance energy transfer. J. Biol.Chem. 277::44925-44931.
Hebert, T.E. et al. 1996. . A peptide derived from a 2-adrenergic receptor transmembrane domain inhibits both receptor dimerization and activation. J. Biol. Chem. 271::16384-16392.
Salahpour, A. et al. 2004. . Homodimerization of the 2-adrenergic receptor as a prerequisite for cell surface targeting. J. Biol. Chem. 279::33390-33397.
Jordan, B.A., Trapaidze, N., Gomes, I., Nivarthi, R., and Devi, L.A. 2001. . Oligomerization of opioid receptors with 2-adrenergic receptors: a role in trafficking and mitogen-activated protein kinase activation. Proc. Natl. Acad. Sci. U. S. A. 98::343-348.
Hague, C. et al. 2006. . Heterodimers of 1B- and 1D-adrenergic receptors form a single functional entity. Mol. Pharmacol. 69::45-55.
Uberti, M.A., Hague, C., Oller, H., Minneman, K.P., and Hall, R.A. 2005. . Heterodimerization with 2-adrenergic receptors promotes surface expression and functional activity of 1D-adrenergic receptors. J. Pharmacol. Exp. Ther. 313::16-23.
Breit, A., Lagace, M., and Bouvier, M. 2004. . Hetero-oligomerization between 2- and 3-adrenergic receptors generates a -adrenergic signaling unit with distinct functional properties. J. Biol. Chem. 279::28756-28765.
Barki-Harrington, L., Luttrell, L.M., and Rockman, H.A. 2003. . Dual inhibition of -adrenergic and angiotensin II receptors by a single antagonist: a functional role for receptor-receptor interaction in vivo. Circulation. 108::1611-1618.
Grandordy, B.M., Mak, J.C.W., and Barnes, P.J. 1994. . Modulation of airway smooth muscle -receptor function by a muscarinic agonist. Life Sci. 54::185-191.
McGraw, D.W. et al. 2006. . Airway smooth muscle prostaglandin-EP1 receptors directly modulate 2–adrenergic receptors within a unique heterodimeric complex. J. Clin. Invest. 116::1400-1409. doi:10.1172/JCI25840.
Barnes, P.J. 1998. . Pharmacology of airway smooth muscle. Am. J. Respir. Crit. Care Med. 158::S123-S132.
Lee, B. et al. 1998. . Influence of the CCR2-V64I polymorphism on human immunodeficiency virus type 1 coreceptor activity and on chemokine receptor function of CCR2b, CCR3, CCR5, and CXCR4. J. Virol. 72::7450-7458..(Peter J. Barnes)
Abstract
Most G protein–coupled receptors (GPCRs) probably exist as homodimers, but it is increasingly recognized that GPCRs may also dimerize with other types of GPCRs and that this physical interaction may affect the function of either receptor. A study in this issue of the JCI demonstrates how heterodimerization between prostaglandin E receptors and 2–adrenergic receptors (2ARs) in airway smooth muscle cells results in uncoupling of 2ARs and a diminished bronchodilator response to 2AR agonists. This illustrates what we believe to be a novel mechanism of receptor cross-talk and highlights the potential importance of GPCR heterodimerization in diseases such as asthma and how this could lead to the development of more specific therapies in the future.
See the related article beginning on page 1400
Cross-talk between different receptors has long been recognized as an important determinant of cellular response in health and disease. Traditionally this cross-talk has been explained by interaction of intracellular signal transduction pathways, phosphorylation of receptors and regulatory proteins by kinases, or effects on intracellular calcium release (1). Receptor cross-talk represents a means of fine-tuning the control of cellular function and is relevant to understanding disease and response to therapeutic agents that interact with cell-surface receptors. Recently there has been growing recognition that physical interaction between cell surface receptors may be a novel means of receptor cross-talk, and this has been studied in the greatest detail for G protein–coupled receptors (GPCRs).
Approximately 400 GPCRs are known to mediate the effects of endogenous ligands and are the targets for about half of currently used prescription drugs (2-4). The interaction of an agonist with the binding pocket of a GPCR induces a conformational change in the transmembrane-spanning segments. This results in its association with a G protein that leads to activation of a signal transduction pathway, resulting in the characteristic cellular response. GPCRs were conventionally thought to exist and act as monomers, but there is accumulating evidence that most GPCRs probably exist as dimers or even oligomers (5-7). Furthermore, different GPCRs may interact with each other, forming heterodimers. This has important implications for understanding cellular regulation and the action of agonists. Dimerization of GPCRs was first proposed by Agnati and colleagues in the 1980s (8), based on the finding of unexplained cooperativity between certain agonists and a larger-than-expected molecular size of receptor proteins observed by gel electrophoresis. However, this idea received little attention until the last decade.
Receptor homodimerization
Although the idea of receptor dimerization was at first resisted and then thought to be the exception or a feature of artificial overexpression systems, it is now clear that most GPCRs exist as homodimers for at least some period during their existence. This has most convincingly been demonstrated using fluorescence resonance energy transfer or bioluminescence resonance energy transfer (BRET), which directly demonstrate receptor protein dimerization in living cells. Indeed, studies using BRET indicate that more than 80% of 2–adrenergic receptors (2ARs) exist as homodimers (9). This dimerization appears to occur during the synthesis of receptors in the endoplasmic reticulum and is necessary for the transport of the newly formed receptors to the cell surface (5-7). Furthermore, interfering with receptor homodimerization affects the trafficking and function of the receptor (7). The sites of physical interaction between GPCRs appear to be the transmembrane -helices. Using a peptide that corresponds to transmembrane helix VI of the 2AR in order to interfere with the presumed dimerization sequence, there was a significant reduction in the incidence of dimerization, and this was associated with reduced activation of adenylyl cyclase by 2AR agonists (10). This suggests that high-affinity binding of receptor and G protein may require the GPCR dimer. Furthermore, mutation of the putative dimerization motif in helix VI prevented the movement of 2ARs from their site of synthesis in the endoplasmic reticulum to the cell surface, indicating that homodimerization is critical for cell-surface expression of 2ARs (11). There is debate about the effect of agonists on the extent of receptor dimerization, but most studies indicate that dimers are formed during receptor synthesis in the endoplasmic reticulum and are formed in the absence of agonist stimulation (7).
Receptor heterodimerization
There is increasing evidence that different GPCRs may form heterodimers and that this can affect the function of each agonist, resulting in significant functional interactions. Indeed, this phenomenon may account for some drug interactions that were unexpected or previously difficult to explain. Many different GPCR heterodimers have now been described (5-7), but the functional consequence of heterodimerization is not predictable. 2ARs may interact with both - and -opioid receptors. This does not appear to affect the binding or effects of agonists but has an effect on receptor trafficking (12). When 2ARs are coexpressed with -opioid receptors, both a 2AR-agonist and a -opioid agonist cause downregulation and internalization of the -opioid receptor, whereas when coexpressed with -opioid receptors, neither agonist causes receptor internalization (12). The 1D–adrenergic receptor is not normally expressed on the cell surface of most cells unless it dimerizes with 1B receptors or 2ARs, indicating an important role for heterodimerization in the trafficking of certain receptors from the endoplasmic reticulum to the cell surface (13, 14). A particular GPCR can only dimerize with certain other GPCRs; receptors that are closely related in structure in general have a higher affinity for interaction, so that homodimers form more readily than heterodimers. However, 2ARs may heterodimerize with 1ARs and 3ARs with a similar affinity to themselves, which suggests that at equal levels of expression, an equal number of homodimers and heterodimers is likely to exist (9, 15). There is still little known about how heterodimerization affects receptor function and signaling. Heterodimerization between 2ARs and angiotensin II type 1 (AT1) receptors appears to account for the crossover effects of beta blockers and AT1 receptor blockers on myocardial function. A beta blocker reduces the effect of angiotensin II in murine cardiomyocytes in vitro and on cardiac function in vivo, whereas an AT1 receptor blocker inhibits the response to a -agonist (16).
Receptor heterodimerization in airway smooth muscle
Airway smooth muscle tone is regulated by multiple GPCRs, and cross-talk between different classes of receptor, such as 2ARs and muscarinic M3 receptors, has previously been demonstrated (17). These interactions may be of clinical relevance in asthma, where airway smooth muscle tone is increased. In this issue of the JCI, McGraw and colleagues demonstrate a novel type of cross-talk between GPCRs involving receptor heterodimerization and demonstrate its functional consequences on murine airway smooth muscle contraction (18). PGE2 activated the PGE2 receptor, EP1 subtype (EP1R) on airway smooth muscle cells, which is coupled to Gq and calcium ion release and yet did not elicit contraction as expected. However, PGE2 reduced the bronchodilator response to a 2AR agonist by attenuating the increase in cAMP. Forskolin, a direct activator of adenylyl cyclase, was unaffected, indicating an upstream interaction between PGE2 and 2AR agonist. Using fluorescence microscopy in airway smooth muscle cells, and BRET and coimmunoprecipitation in a cell line, heterodimerization between EP1Rs and 2ARs was demonstrated. EP1R agonists bind to the heterodimer and uncouple 2ARs from Gs, thus diminishing the bronchodilator response of the 2AR agonist (Figure 1). This represents a novel level of functional antagonism between bronchoconstrictor and bronchodilator mechanisms and may contribute to the reduced response to 2AR agonists that may occur in severe asthma, when endogenous concentrations of PGE2 may be elevated. Since airway smooth muscle cells express more than 30 different GPCRs, many other possible GPCR heterodimerizations remain to be explored (19).
Clinical implications
At present there is relatively little information about the functional consequences of receptor heterodimerization, but the study by McGraw et al. (18) demonstrates that such receptor interactions may have important functional consequences. Receptor heterodimerization may affect the surface expression of receptors, the rate of receptor desensitization, and the effect of agonists on signal transduction, resulting in several different and so-far unpredictable functional consequences. This allows for the possibility of finding unexpected drug interactions or novel therapeutic agents that selectively activate certain heterodimer pairs. Since the relative expression of different GPCRs in various cell types differs, this makes it potentially possible to develop more selective drugs in the future. The role of receptor heterodimerization in disease has hardly been explored, but genetic polymorphisms in areas of the receptor that affect dimerization with other receptors may alter the function of the receptor, as has already been demonstrated for chemokine receptors (6, 20). GPCR heterodimerization appears to be a novel means of cell regulation that is likely to have clinical and therapeutic significance in the future.
Footnotes
Nonstandard abbreviations used: 2AR, 2–adrenergic receptor; AT1, angiotensin II type 1; BRET, bioluminescence resonant energy transfer; EP1R, PGE2 receptor, EP1 subtype; GPCR, G protein–coupled receptor.
Conflict of interest: The author has declared that no conflict of interest exists.
References
Vazquez-Prado, J., Casas-Gonzalez, P., and Garcia-Sainz, J.A. 2003. . G protein-coupled receptor cross-talk: pivotal roles of protein phosphorylation and protein-protein interactions. Cell. Signal. 15::549-557.
Drews, J. 1996. . Genomic sciences and the medicine of tomorrow. Nat. Biotechnol. 14::1516-1518.
Pierce, K.L., Premont, R.T., and Lefkowitz, R.J. 2002. . Seven-transmembrane receptors. Nat. Rev. Mol. Cell Biol. 3::639-650.
Hill, S.J. 2006. . G-protein-coupled receptors: past, present and future. Br. J. Pharmacol. 147(Suppl. 1)::S27-S37.
Milligan, G. 2004. . G protein-coupled receptor dimerization: function and ligand pharmacology. Mol. Pharmacol. 66::1-7.
Prinster, S.C., Hague, C., and Hall, R.A. 2005. . Heterodimerization of G protein-coupled receptors: specificity and functional significance. Pharmacol. Rev. 57::289-298.
Bulenger, S., Marullo, S., and Bouvier, M. 2005. . Emerging role of homo- and heterodimerization in G-protein-coupled receptor biosynthesis and maturation. Trends Pharmacol. Sci. 26::131-137.
Agnati, L.F., Fuxe, K., Zoli, M., Rondanini, C., and Ogren, S.O. 1982. . New vistas on synaptic plasticity: the receptor mosaic hypothesis of the engram. Med. Biol. 60::183-190.
Mercier, J.F., Salahpour, A., Angers, S., Breit, A., and Bouvier, M. 2002. . Quantitative assessment of 1- and 2-adrenergic receptor homo- and heterodimerization by bioluminescence resonance energy transfer. J. Biol.Chem. 277::44925-44931.
Hebert, T.E. et al. 1996. . A peptide derived from a 2-adrenergic receptor transmembrane domain inhibits both receptor dimerization and activation. J. Biol. Chem. 271::16384-16392.
Salahpour, A. et al. 2004. . Homodimerization of the 2-adrenergic receptor as a prerequisite for cell surface targeting. J. Biol. Chem. 279::33390-33397.
Jordan, B.A., Trapaidze, N., Gomes, I., Nivarthi, R., and Devi, L.A. 2001. . Oligomerization of opioid receptors with 2-adrenergic receptors: a role in trafficking and mitogen-activated protein kinase activation. Proc. Natl. Acad. Sci. U. S. A. 98::343-348.
Hague, C. et al. 2006. . Heterodimers of 1B- and 1D-adrenergic receptors form a single functional entity. Mol. Pharmacol. 69::45-55.
Uberti, M.A., Hague, C., Oller, H., Minneman, K.P., and Hall, R.A. 2005. . Heterodimerization with 2-adrenergic receptors promotes surface expression and functional activity of 1D-adrenergic receptors. J. Pharmacol. Exp. Ther. 313::16-23.
Breit, A., Lagace, M., and Bouvier, M. 2004. . Hetero-oligomerization between 2- and 3-adrenergic receptors generates a -adrenergic signaling unit with distinct functional properties. J. Biol. Chem. 279::28756-28765.
Barki-Harrington, L., Luttrell, L.M., and Rockman, H.A. 2003. . Dual inhibition of -adrenergic and angiotensin II receptors by a single antagonist: a functional role for receptor-receptor interaction in vivo. Circulation. 108::1611-1618.
Grandordy, B.M., Mak, J.C.W., and Barnes, P.J. 1994. . Modulation of airway smooth muscle -receptor function by a muscarinic agonist. Life Sci. 54::185-191.
McGraw, D.W. et al. 2006. . Airway smooth muscle prostaglandin-EP1 receptors directly modulate 2–adrenergic receptors within a unique heterodimeric complex. J. Clin. Invest. 116::1400-1409. doi:10.1172/JCI25840.
Barnes, P.J. 1998. . Pharmacology of airway smooth muscle. Am. J. Respir. Crit. Care Med. 158::S123-S132.
Lee, B. et al. 1998. . Influence of the CCR2-V64I polymorphism on human immunodeficiency virus type 1 coreceptor activity and on chemokine receptor function of CCR2b, CCR3, CCR5, and CXCR4. J. Virol. 72::7450-7458..(Peter J. Barnes)