Biomedical subjects

Jakob Lerche Hansen

Publications and source records attributed to Jakob Lerche Hansen.

9 recordsLinked to original sources

PubMed2006

Constitutive homo- and hetero-oligomerization of TbetaRII-B, an alternatively spliced variant of the mouse TGF-beta type II receptor.

Transforming growth factor (TGF)-beta ligands signal through transmembrane type I and type II serine/threonine kinase receptors, which form heteromeric signalling complexes upon ligand binding. Type II TGF-beta receptors (TbetaRII) are reported to exist as homodimers at the cell surface, but the oligomerization pattern and dynamics of TbetaRII splice variants in live cells has not been demonstrated thus far. Using co-immunoprecipitation and bioluminescence resonance energy transfer (BRET), we demonstrate that the mouse TbetaRII receptor splice variant TbetaRII-B is capable of forming ligand-independent homodimers and heterodimers with TbetaRII. The homomeric interaction of mouse (m)TbetaRII-B isoforms, however, is less robust than the heteromeric interactions of mTbetaRII-B with wild-type TbetaRII, which indicates that these receptors may be more likely to heterodimerize when both receptors are expressed. Moreover, we demonstrate that mTbetaRII-B is a signalling receptor with ubiquitous tissue expression. Our study thus demonstrates previously unappreciated complex formation of TGF-beta type II receptors, and suggests that mTbetaRII-B can direct TGF-beta-induced signalling in vitro and in vivo.

Amino Acid Sequence↗

PubMed2006

Extracellular calcium sensing in rat aortic vascular smooth muscle cells.

Extracellular calcium (Ca(2+)(o)) can act as a first messenger in many cell types through a G protein-coupled receptor, calcium-sensing receptor (CaR). It is still debated whether the CaR is expressed in vascular smooth muscle cells (VSMCs). Here, we report the expression of CaR mRNA and protein in rat aortic VSMCs and show that Ca(2+)(o) stimulates proliferation of the cells. The effects of Ca(2+)(o) were attenuated by pre-treatment with MAPK kinase 1 (MEK1) inhibitor, as well as an allosteric modulator, NPS 2390. Furthermore, stimulation of the VSMCs with Ca(2+)(o)-induced phosphorylation of ERK1/2, but surprisingly did not cause inositol phosphate accumulation. We were not able to conclusively state that the CaR mediates Ca(2+)(o)-induced cell proliferation. Rather, an additional calcium-sensing mechanism may exist. Our findings may be of importance with regard to atherosclerosis, an inflammatory disease characterized by abnormal proliferation of VSMCs and high local levels of calcium.

Animals↗

PubMed2006

Role of G-protein-coupled receptor kinase 2 in the heart--do regulatory mechanisms open novel therapeutic perspectives?

G-protein-coupled receptor kinase (GRK) 2 regulates a plethora of cellular processes, including cardiac expression and function of key seven-transmembrane receptors (7TM receptors) such as the beta-adrenergic and angiotensin receptors (Penela P, Murga C, Ribas C, et al.: 2006. Mechanisms of regulation of G-protein-coupled receptor kinases [GRKs] and cardiovascular disease. Cardiovasc Res 69:46-56, Rockman HA, Koch WJ, Lefkowitz RJ: 2002. Seven-transmembrane-spanning receptors and heart function. Nature 415:206-212). Interestingly, these two G-protein-coupled receptor systems are targeted by modern heart failure treatment including beta-adrenergic blockers, angiotensin-converting enzyme inhibitors, and angiotensin receptor blockers. Although GRK2 is ubiquitously expressed, its particular importance in the heart has been demonstrated by interesting phenotypes of genetically altered mice that suggest GRK2 inhibition can ameliorate heart failure. In essence, this work suggests GRK2 could be an endogenous receptor blocker targeting both the beta-adrenergic and angiotensin receptors in the heart. This notion immediately suggests it is important to understand the molecular mechanisms that regulate GRK2 activity in the heart. In this review, we provide a detailed presentation of the tight regulation of GRK2 expression levels and protein activity, and we discuss the cardiovascular GRK2 functions and possible therapeutic perspectives.

Animals↗

PubMed2006

Family C 7TM receptor dimerization and activation.

The family C seven transmembrane (7TM) receptors constitutes a small and especially well characterized subfamily of the large 7TM receptor superfamily. Approximately 50% of current prescription drugs target 7TM receptors, this biologically important family represents the largest class of drug-targets today. It is well established that family C 7TM receptors form homo- or hetero-dimers on the cell surface of living cells. The large extra-cellular domains (ECD) have been crystallized as a dimer in the presence and absence of agonist. Upon agonist binding, the dimeric ECD undergoes large conformational changes that lead to receptor activation. Despite extensive studies of the receptor transmembrane domain, several key features, including the exact organization of the complete receptor dimer, the sequence of events leading to receptor activation, and the functional significance of dimerization, have yet to be fully defined. This review presents the biochemical support for family C 7TM receptor dimerization and discusses its importance for receptor biosynthesis, surface expression, ligand binding and activation, since lessons learnt here may well be applicable to the whole superfamily of 7TM receptors.

Animals↗

PubMed2005

Calcium receptor is functionally expressed in rat neonatal ventricular cardiomyocytes.

Both intra- and extracellular calcium play multiple roles in the physiology and pathophysiology of cardiomyocytes, especially in stimulus-contraction coupling. The intracellular calcium level is closely controlled through the concerted actions of calcium channels, exchangers, and pumps; however, the expression and function(s) of the so-called calcium-sensing receptor (CaR) in the heart remain less well characterized. The CaR is a seven-transmembrane receptor, which, in response to noncovalent binding of extracellular calcium, activates intracellular effectors, including G proteins and extracellular signal-regulated kinases (ERK1/2). We have shown that cultured neonatal cardiomyocytes express the CaR messenger RNA and the CaR protein. Furthermore, increasing concentrations of extracellular calcium and a type II CaR activator "calcimimetic" caused inositol phosphate (IP) accumulation, downregulated tritiated thymidine incorporation, and supported ERK1/2 phosphorylation, suggesting that the CaR protein is functionally active. Interestingly, the calcimimetic induced a more rapid ERK1/2 phosphorylation than calcium and left-shifted the IP concentration-response curve for extracellular calcium, supporting the hypothesis that CaR is functionally expressed in cardiac myocytes. This notion was underscored by studies using a virus containing a dominant-negative CaR construct, because this protein blunted the calcium-induced IP response. In conclusion, we have shown that the CaR is functionally expressed in neonatal ventricular cardiomyocytes and that the receptor activates second messenger pathways, including IP and ERK, and decreases DNA synthesis. A specific calcium-sensing receptor on cardiac myocytes could play a role in regulating cardiac development, function, and homeostasis.

Animals↗

PubMed2004

Oligomerization of wild type and nonfunctional mutant angiotensin II type I receptors inhibits galphaq protein signaling but not ERK activation.

The 7-transmembrane or G protein-coupled receptors relay signals from hormones and sensory stimuli to multiple signaling systems at the intracellular face of the plasma membrane including heterotrimeric G proteins, ERK1/2, and arrestins. It is an emerging concept that 7-transmembrane receptors form oligomers; however, it is not well understood which roles oligomerization plays in receptor activation of different signaling systems. To begin to address this question, we used the angiotensin II type 1 (AT(1)) receptor, a key regulator of blood pressure and fluid homeostasis that in specific context has been described to activate ERKs without activating G proteins. By using bioluminescence resonance energy transfer, we demonstrate that AT(1) receptors exist as oligomers in transfected COS-7 cells. AT(1) oligomerization was both constitutive and receptor-specific as neither agonist, antagonist, nor co-expression with three other receptors affected the bioluminescence resonance energy transfer 2 signal. Furthermore, the oligomerization occurs early in biosynthesis before surface expression, because we could control AT(1) receptor export from the endoplasmic reticulum or Golgi by using regulated secretion/aggregation technology (RPD trade mark ). Co-expression studies of wild type AT(1) and AT(1) receptor mutants, defective in either ligand binding or G protein and ERK activation, yielded an interesting result. The mutant receptors specifically exerted a dominant negative effect on Galpha(q) activation, whereas ERK activation was preserved. These data suggest that distinctly active conformations of AT(1) oligomers can couple to each of these signaling systems and imply that oligomerization plays an active role in supporting these distinctly active conformations of AT(1) receptors.

Animals↗

PubMed2004

Functional consequences of 7TM receptor dimerization.

7TM receptors work as signaling platforms that activate multiple signalling systems at the intracellular face of the plasma membrane. It is an emerging concept that 7TM receptors form homo- and hetero-dimers or -oligomers in vitro and in vivo. Numerous studies suggest dimerization is important for receptor function including agonist/antagonist affinity, efficacy, trafficking, and specificity of signal transduction, yet it remains unknown whether dimerization is a prerequisite for 7TM receptor signaling. The current review provides an overview of the biochemical support for 7TM homodimerization, followed by a discussion of the characteristics of homodimerization, with focus on dimer organization, and the functional consequences of dimerization. Heterodimerization will not generally be discussed in this review although we have included a few examples to illustrate specific points, and a table that summarises the current literature on this subject.

Animals↗

PubMed2004

Loss-of-function polymorphic variants of the human angiotensin II type 1 receptor.

The angiotensin II type 1 (AT1) receptor is the primary effector for angiotensin II (Ang II), a key peptide regulator of blood pressure and fluid homeostasis. AT1 receptors are involved in the pathogenesis of several cardiovascular diseases, including hypertension, cardiac hypertrophy, and congestive heart failure, which are characterized by significant interindividual variation in disease risk, progression, and response to pharmacotherapy. Such variation could arise from genomic polymorphisms in the AT1 receptor. To pursue this notion, we have pharmacologically characterized seven known and putative nonsynonymous AT1 receptor variants. Functional analysis using the cell-based assay receptor selection and amplification technology (R-SAT) revealed that three variants (AT1-G45R, AT1-F204S, and AT1-C289W) displayed altered responses to Ang II and other AT1 receptor agonists and antagonists. Agonist responses to Ang II were absent for AT1-G45R and significantly reduced in potency for AT1-C289W (11-fold) and AT1-F204S (57-fold) compared with the wild-type (WT) receptor. AT1-F204S also displayed reduced relative efficacy (57%). Quantitatively similar results were obtained in two additional functional assays, phosphatidyl inositol hydrolysis and extracellular signal-regulated kinase activation. Radioligand binding studies revealed that AT1-G45R failed to bind Ang II, whereas cell surface staining clearly showed that it trafficked to the cell surface. AT1-C289W and AT1-F204S displayed reduced binding affinities of 3- and 5-fold and reduced cell surface expression of 43 and 60% of that observed for the WT receptor, respectively. These data demonstrate that polymorphic variation in the human AT1 receptor induces loss of functional phenotypes, which may constitute the molecular basis of variability of AT1 receptor-mediated physiological responses.

Amino Acid Sequence↗

PubMed2002

Extracellular signal-regulated kinases control expression of G protein-coupled receptor kinase 2 (GRK2).

G protein-coupled receptor kinase 2 (GRK2) phosphorylates G protein-coupled receptors resulting in uncoupling from G proteins. Receptors modulate GRK2 expression, however the mechanistic basis for this effect is largely unknown. Here we report a novel mechanism by which receptors use the extracellular signal-regulated kinase (ERK) cascade to regulate GRK2 cellular levels. ERK activation by receptor stimulation elevated endogenous GRK2 while antagonist treatment decreased cellular GRK2. Activating ERK by overexpressing constitutive active MEK-1 or Ras elevated GRK2 protein levels while blocking ERK using PD98059 or dominant negative Ras abolished this effect. These data suggest ERK is a critical regulator of GRK2 levels.

Animals↗