Patrick Tarnow

Mutually Opposite Signal Modulation by Hypothalamic Heterodimerization of Ghrelin and Melanocortin-3 Receptors

Background: The melanocortin-3 (MC3R) and ghrelin (GHSR) receptors are important key components in hypothalamic weight regulation. Results: MC3R and GHSR di/oligomerize and have an opposite impact on each others function. Conclusion: The high basal activity of GHSR is a determinant of heterodimer function, and MC3R may constrain GHSR function. Significance: Receptor di/oligomerization and its functional relevance contribute to the complex network of hypothalamic weight regulation. Interaction and cross-talk of G-protein-coupled receptors (GPCRs) are of considerable interest because an increasing number of examples implicate a profound functional and physiological relevance of homo- or hetero-oligomeric GPCRs. The ghrelin (growth hormone secretagogue receptor (GHSR)) and melanocortin-3 (MC3R) receptors are both known to have orexigenic effects on the hypothalamic control of body weight. Because in vitro studies indicate heterodimerization of GHSR and MC3R, we investigated their functional interplay. Combined in situ hybridization and immunohistochemistry indicated that the vast majority of GHSR-expressing neurons in the arcuate nucleus also express MC3R. In vitro coexpression of MC3R and GHSR promoted enhanced melanocortin-induced intracellular cAMP accumulation compared with activation of MC3R in the absence of GHSR. In contrast, agonist-independent basal signaling activity and ghrelin-induced signaling of GHSR were impaired, most likely due to interaction with MC3R. By taking advantage of naturally occurring GHSR mutations and an inverse agonist for GHSR, we demonstrate that the observed enhanced MC3R signaling capability depends directly on the basal activity of GHSR. In conclusion, we demonstrate a paradigm-shifting example of GPCR heterodimerization allowing for mutually opposite functional influence of two hypothalamic receptors controlling body weight. We found that the agonist-independent active conformation of one GPCR can determine the signaling modalities of another receptor in a heterodimer. Our discovery also implies that mutations within one of two interacting receptors might affect both receptors and different pathways simultaneously. These findings uncover mechanisms of important relevance for pharmacological targeting of GPCR in general and hypothalamic body weight regulation in particular.

Heterodimerization of Hypothalamic G-Protein-Coupled Receptors Involved in Weight Regulation

Background: Melanocortin 3 and 4 receptors (MC3R and MC4R) are known to play an essential role in hypothalamic weight regulation. In addition to these two G-protein-coupled receptors (GPCRs), a huge number of other GPCRs are expressed in hypothalamic regions, and some of them are involved in weight regulation. So far, homodimerization was shown for a few of these receptors. Heterodimerization of unrelated receptors may have profound functional consequence but heterodimerization of GPCRs involved in weight regulation was not reported yet. Methods: A selective number of hypothalamically expressed GPCRs were cloned into a eukaryotic expression vector. Cell surface expression was demonstrated by an ELISA approach. Subcellular distribution was investigated by confocal laser microscopy. A sandwich ELISA and fluorescence resonance energy transfer (FRET) were used to determine protein-protein interaction. Results: Via sandwich ELISA and FRET approach we could demonstrate a robust interaction of the MC4R with GPR7, both of which are expressed in the hypothalamic nucleus paraventricularis. Moreover, we determined a strong interaction of MC3R with the growth hormone secretagogue receptor expressed in the nucleus arcuatus. Conclusion: Identification GPCR heterodimerization adds to the understanding of the complexity of weight regulation and may provide important information to develop therapeutic strategies to treat obesity.

A heterozygous mutation in the third transmembrane domain causes a dominant-negative effect on signalling capability of the MC4R.

Background: Heterozygous MC4R mutation is the most frequent cause of monogenic obesity. For most MC4R mutations a gene dosage effect seems to be the underlying mechanism. However, a dominant negative effect of a heterozygous MC4R mutation was recently identified, pointing to an additional mechanism of MC4R inactivation. Methods: The complete loss-of-function mutation (Ser136Phe), identified in a cohort of obese Austrian patients, was characterized for cell surface expression, signal transduction and ligand binding properties. Co-transfection studies tested for a dominant negative effect. Dimerization was investigated by a sandwich ELISA and by fluorescence resonance energy transfer (FRET) approach. Potential intramolecular interactions of Ser136 were studied by homologous receptor modelling based on the crystal structure of the β2-adrenergic receptor. Results: The Ser136Phe mutation showed a dominant negative effect. The sandwich ELISA and FRET approach demonstrated dimerization of mutant and wild type receptor. Receptor modelling revealed an essential function of Ser136 at transmembrane helix 3 (TMH3) for establishing H-bonds between TMH2, TMH3, and TMH7. The mutation Ser136Phe most likely disrupts this network and leads to an incompetent helix-helix arrangement in the mutated receptor. Conclusion: Identification of dominant negative MC4R mutations is important to fully understand receptor function and to determine receptor regions that are involved in MC4R dimer activation.

Inhibition of melanocortin-4 receptor dimerization by substitutions in intracellular loop 2.

Obesity is one of the most challenging global health problems. One key player in energy homeostasis is the melanocortin-4 receptor (MC4R), which is a family A G-protein-coupled receptor (GPCR). It has recently been shown that MC4R has the capacity to form homo- or heterodimers. Dimerization of GPCRs is of great importance for signaling regulation, with major pharmacological implications. Unfortunately, not enough is yet known about the detailed structural properties of MC4R dimers or the functional consequences of receptor dimerization. Our goal, therefore, was to explore specific properties related to MC4R dimerization. First, we aimed to induce the dissociation of dimers to monomers and to compare the functional parameters of wild-type and MC4R variants. To inhibit homodimerization, we designed MC4R chimeras with the cannabinoid-1 receptor, a receptor that does not interact with MC4R. Indeed, we identified several substitutions in the intracellular loop 2 (ICL2) and adjacent regions of transmembrane helix 3 (TMH3) and TMH4 that lead to partial dimer dissociation. Interestingly, the capacity for signaling activity was generally increased in these MC4R variants, although receptor expression remained unchanged. This increase in activity for dissociated receptors might indicate a link between receptor dimerization and signaling capacity. Moreover, dimer dissociation was also observed in a naturally occurring activating MC4R mutation in ICL2. Taken together, this study provides new information on the structural prerequisites for MC4R dimerization and identifies an approach to induce the dissociation of MC4R dimers. This might be useful for further investigation of pharmacological properties.

MC4R oligomerizes independently of extracellular cysteine residues

The melanocortin 4 receptor (MC4R) plays an essential role in weight regulation. Recently we could show that the MC4R is able to form receptor dimers. In the present study we investigated the role of extracellular cysteine residues and the structure of the third extracellular loop for receptor dimerization. None of the four extracellular cysteine residues nor the structure of the third extracellular loop play a role for MC4R-MC4R interaction as all investigated mutants display the same dimerization pattern as the wild-type receptor. Therefore for MC4R dimerization structures of the transmembrane-spanning helices are more likely to be involved.

A New Phenotype of Nongoitrous and Nonautoimmune Hyperthyroidism Caused by a Heterozygous Thyrotropin Receptor Mutation in Transmembrane Helix 6

CONTEXT Activating mutations in the TSHR gene were found in patients suffering from nonautoimmune hyperthyroidism. In the past, it was assumed that thyroid hyperplasia is due to constitutive activation of the Gs/adenylyl cyclase signaling pathway; however, the physiological role of the Gq/11 pathway in this context remains unclear. OBJECTIVE In this study, we investigated molecular details of the TSHR in a patient with nonautoimmune and nongoitrous hyperthyroidism. RESULTS We detected a heterozygous mutation in exon 10 of the TSHR gene leading to an exchange of a cysteine residue for tryptophan at amino acid position 636 in transmembrane helix 6. Functional characterization of the mutant receptor revealed a slight reduction of the cell surface expression and TSH induced cAMP accumulation compared to the wild type. Additional observations included a constitutive activation of the Gs-mediated signaling pathway and a simultaneous nearly complete loss-of-function for the Gq/11 pathway after bovine TSH stimulation. Studies on TSHR models suggest significant changes of important amino acid interactions and the overall helix arrangement caused by mutation C636W. CONCLUSION We report a patient in whom a TSHR mutation leads to nonautoimmune hyperthyroidism due to a mutation that constitutively activates the Gs signaling pathway but additionally completely inhibits the Gq/11 pathway. The absence of goiter in the patient suggests that the Gq/11 pathway is related to thyroid growth and that different signaling pathways are mediated and regulated by TSH. These functional data could be confirmed by reproducible findings of two siblings with a constitutive activation for both pathways.

Identification of the translation start site of the human melanocortin 3 receptor.

Background: The melanocortin-3-receptor (MC3R) is a G-protein coupled receptor participating in hypothalamic energy metabolism. So far, it was assumed that the translation of the human MC3R starts at the non-conserved first ATG, however, a second evolutionary conserved ATG is located 37 amino acids downstream. One frequent polymorphism, T6K, is located between these two ATGs. Methods: For characterization of the two potential start ATGs, COS-7 cells were transfected with plasmids encoding the longer and the shorter form of the human MC3R. For signal transduction properties, cAMP was measured. Cell surface expression was determined by using an ELISA method. The translational start point of the MC3R was investigated by a GFP-based method. Results: Signal transduction was comparable for the long and the short receptor form. Cell surface expression via aminoterminal hemagglutinin tag could only be detected in the shorter form, but not in the longer one. In our study we show that the translation of the human MC3R protein starts at the evolutionary conserved ATG codon which results in a shorter protein than previously assumed. Conclusion: The polymorphism T6K is not located in the coding region of the human MC3R and has no influence on translation initiation which makes an impact on body weight unlikely.