Heptahelical receptors (HHRs) are generally thought to function as monomeric entities. Several HHRs such as somatostatin receptors (SSTRs), however, form homo-and heterooligomers when activated by ligand binding. By using dual fluorescent ligands simultaneously applied to live cells monotransfected with SSTR5 (R5) or SSTR1 (R1), or cotransfected with R5 and R1, we have analyzed the ligand receptor stoichiometry and aggregation states for the three receptor systems by fluorescence resonance energy transfer and fluorescence correlation spectroscopy. Both homo-and heterooligomeric receptors are occupied by two ligand molecules. We find that monomeric, homooligomeric, and heterooligomeric receptor species occur in the same cell cotransfected with two SSTRs, and that oligomerization of SSTRs is regulated by ligand binding by a selective process that is restricted to some (R5) but not other (R1) SSTR subtypes. We propose that induction by ligand of different oligomeric states of SSTRs represents a unique mechanism for generating signaling specificity not only within the SSTR family but more generally in the HHR family. H eptahelical receptors (HHRs) constitute the largest single family of transmembrane signaling molecules that respond to diverse external stimuli such as hormones, neurotransmitters, chemoattractants, odorants, and photons. Although these receptors have been generally thought to function as monomeric entities, there is growing evidence that a number of HHRs assemble as functional homo-and heterooligomers (1, 2). Dimerization seems to be necessary for function of the class C subfamily of HHRs comprising the metabotropic glutamate, calcium sensing, the GABAB, and pheromone receptors that are targeted to the plasma membrane as preformed dimers which are stabilized by ligand binding (3-7). Several HHRs such as somatostatin receptors (SSTRs), dopamine receptors, gonadotrophin-releasing hormone receptor (GnRHR), luteinizing hormone͞chorionic gonadotrophin hormone receptor, and chemokine receptors, however, which belong to the rhodopsin-like class A subfamily of HHRs, assemble on the membrane as homo-and heterooligomeric species in response to agonist activation (8-15).In the case of SSTRs, we have shown by photobleaching fluorescence resonance energy transfer (pbFRET) that the human (h) type 5 receptor (hSSTR5 or R5) exists in the basal state as a monomer, and that activation by ligand induces dose-dependent oligomerization (8). When coexpressed with another SSTR (hSSTR1 or R1) or an unrelated HHR such as the dopamine 2 receptor (D 2 R), R5 also forms a heterooligomer that displays pharmacological properties distinct from those of either of the separate receptors (9). Little is known about the stoichiometry of ligand-receptor reactions or the specificity for homoand heterooligomeric interactions between two receptors that are coexpressed in the same cell. By using dual fluorescent ligands simultaneously applied to live cells monotransfected with R5 or R1, or cotransfected with R5 and R1, we have analyzed the ...

Calcium-sensing receptors (CaSRs) regulate systemic calcium homeostasis in the parathyroid gland, kidney, intestine, and bone and translate fluctuations in serum calcium into peptide hormone secretion, cell signaling, and regulation of gene expression. The CaSR is a G protein (heterotrimeric guanosine triphosphate-binding protein)-coupled receptor that operates in the constant presence of agonist, sensing small changes with high cooperativity and minimal functional desensitization. Here, we used multiwavelength total internal reflection fluorescence microscopy to demonstrate that the signaling properties of the CaSR result from agonist-driven maturation and insertion of CaSRs into the plasma membrane. Plasma membrane CaSRs underwent constitutive endocytosis without substantial recycling, indicating that signaling was determined by the rate of insertion of CaSRs into the plasma membrane. Intracellular CaSRs colocalized with calnexin in the perinuclear endoplasmic reticulum and formed complexes with 14-3-3 proteins. Ongoing CaSR signaling resulted from agonist-driven trafficking of CaSR through the secretory pathway. The intracellular reservoir of CaSRs that were mobilized by agonist was depleted when glycosylation of newly synthesized receptors was blocked, suggesting that receptor biosynthesis was coupled to signaling. The continuous, signaling-dependent insertion of CaSRs into the plasma membrane ensured a rapid response to alterations in the concentrations of extracellular calcium or allosteric agonist despite ongoing desensitization and endocytosis. Regulation of CaSR plasma membrane abundance represents a previously unknown mechanism of regulation that may be relevant to other receptors that operate in the chronic presence of agonist.

Somatostatin (SST) analogs have been successfully used in the medical treatment of acromegaly, caused by GH hypersecreting pituitary adenomas. Patients on SST analogs rarely develop tachyphylaxis despite years of continuous administration. It has been recently proposed that a functional association between SST receptor (SSTR) subtypes 2 and 5 exists to account for this behavior; however, a physical interaction has yet to be identified. Using both coimmunoprecipitation and photobleaching fluorescence resonance energy transfer microscopy techniques, we determined that SSTR2 and SSTR5 heterodimerize. Surprisingly, selective activation of SSTR2 and not SSTR5, or their costimulation, modulates the association. The SSTR2-selective agonist L-779,976 is more efficacious at inhibiting adenylate cyclase, activating ERK1/2, and inducing the cyclin-dependent kinase inhibitor p27(Kip1) in cells expressing both SSTR2 and SSTR5 compared with SSTR2 alone. Furthermore, cell growth inhibition by L-779,976 treatment was markedly extended in coexpressing cells. Trafficking of SSTR2 is also affected upon heterodimerization, an attribute corresponding to modifications in beta-arrestin association kinetics. Activation of SSTR2 results in the recruitment and stable association of beta-arrestin, followed by receptor internalization and intracellular receptor pooling. In contrast, heterodimerization increases the recycling rate of internalized SSTR2 by destabilizing its interaction with beta-arrestin. Given that SST analogs show preferential binding to SSTR2, these data provide a mechanism for their effectiveness in controlling pituitary tumors and the absence of tolerance seen in patients undergoing long-term administration.

G-protein-coupled receptors (GPCRs) represent the largest and most diverse family of cell surface receptors. Several GPCRs have been documented to dimerize with resulting changes in pharmacology. We have previously reported by means of photobleaching fluorescence resonance energy transfer (pbFRET) microscopy and fluorescence correlation spectroscopic (FCS) analysis in live cells, that human somatostatin receptor (hSSTR) 5 could both homodimerize and heterodimerize with hSSTR1 in the presence of the agonist SST-14. In contrast, hSSTR1 remained monomeric when expressed alone regardless of agonist exposure in live cells. In an effort to elucidate the role of ligand and receptor subtypes in heterodimerization, we have employed both pb-FRET microscopy and Western blot on cells stably coexpressing hSSTR1 and hSSTR5 treated with subtypespecific agonists. Here we provide evidence that activation of hSSTR5 but not hSSTR1 is necessary for heterodimeric assembly. This property was also reflected in signaling as shown by increases in adenylyl cyclase coupling efficiencies. Furthermore, receptor Ctail chimeras allowed for the identification of the C-tail as a determinant for dimerization. Finally, we demonstrate that heterodimerization is subtype-selective involving ligand-induced conformational changes in hSSTR5 but not hSSTR1 and could be attributed to molecular events occurring at the C-tail. Understanding the mechanisms by which GPCRs dimerize holds promise for improvements in drug design and efficacy.In recent years, G-protein-coupled receptors (GPCRs), 1 once believed to exist at the plasma membrane as monomers, have been shown to assemble on the membrane as functional homoand heterodimers (1, 2). Dimerization 2 of GPCRs has been shown to affect a multitude of receptor functions including ligand binding, signaling, receptor desensitization, and receptor trafficking (1, 2). The influence of GPCR dimerization was shown to include cellular immunity, neurotransmission (1), taste (3-5), and disease (6). Although the mechanism by which GPCR dimerization occurs remains obscure, one model suggests that ligand binding of cell surface receptors induces a conformational change that favors dimer formation; while the other suggests that dimerization is an exclusive event occurring early on during receptor biogenesis most probably in the ER and is a necessary event for proper receptor trafficking and function.This latter model has been suggested for members of the class C subfamily of GPCRs, which include the GABAergic receptors (7-9), calcium-sensing receptor (10, 11), the metabotropic glutamate receptor (12), and the sweet taste receptors (3-5). However, this paradigm of GPCR assembly is not consistent among the class A/rhodopsin-like family of GPCRs. Several reports have shown that agonist plays an active role in GPCR dimerization at the plasma membrane, suggesting an equilibrium between GPCR dimers/monomers that can be regulated by ligand occupancy. These receptors include the human somatostatin receptors (hSSTRs) (13,14), dopam...

Calcium-sensing receptor (CASR), expressed in parathyroid gland and kidney, is a critical regulator of extracellular calcium homeostasis. This G protein-coupled receptor exists at the plasma membrane as a homodimer, although it is unclear at which point in the biosynthetic pathway dimerization occurs. To address this issue, we have analyzed wild-type and mutant CASRs harboring R66H, R66C or N583X-inactivating mutations identified in familial hypocalciuric hypercalcemia/neonatal severe hyperparathyroid patients, which were transiently expressed in kidney cells. All mutants were deficient in cell signaling responses to extracellular CASR ligands relative to wild-type. All mutants, although as well expressed as wild-type, lacked mature glycosylation, indicating impaired trafficking from the endoplasmic reticulum (ER). Dimerized forms of wild-type, R66H and R66C mutants were present, but not of the N583X mutant. By immunofluorescence confocal microscopy of non-permeabilized cells, although cell surface expression was observed for the wild-type, little or none was seen for the mutants. In permeabilized cells, perinuclear staining was observed for both wild-type and mutants. By colocalization fluorescence confocal microscopy, the mutant CASRs were localized within the ER but not within the Golgi apparatus. By the use of photobleaching fluorescence resonance energy transfer microscopy, it was demonstrated that the wild-type, R66H and R66C mutants were dimerized in the ER, whereas the N583X mutant was not. Hence, constitutive CASR dimerization occurs in the ER and is likely to be necessary, but is not sufficient, for exit of the receptor from the ER and trafficking to the cell surface.

G-protein-coupled receptors (GPCRs) represent the largest and most diverse family of cell surface receptors. Several GPCRs have been documented to dimerize with resulting changes in pharmacology and signaling. We have previously reported, by means of photobleaching fluorescence resonance energy transfer (pbFRET) microscopy and fluorescence correlation spectroscopic analysis in live cells, that human somatostatin receptor (hSSTR) 5 could both homodimerize and heterodimerize with hSSTR1 in the presence of the agonist SST-14. By contrast, hSSTR1 remained monomeric when expressed alone regardless of agonist exposure in live cells. However, the effect of the agonist on other hSSTR members remains unknown. Using pbFRET microscopy and Western blot, we provide evidence for agonist-dependent dissociation of self-associated hSSTR2 stably expressed in CHO-K1 and HEK-293 cells. Furthermore, the dissociation of the hSSTR2 dimer occurred in a concentrationdependent manner. Moreover, blocking receptor dissociation using a cross-linker agent perturbed receptor trafficking. Taking these data together, we suggest that the process of GPCR dimerization may operate differently, even among members of the same family, and that receptor dissociation as well as dimerization may be important steps for receptor dynamics. G-protein-coupled receptors (GPCRs)1 represent ϳ1% of the human genome, an estimate exceeding 800 genes (1). The initial notion that GPCRs are present on the membrane as monomeric entities in a 1:1 stoichiometric ratio with their Gprotein has since been reinterpreted. Several studies have shown by using a combination of techniques such as co-immunoprecipitation, bioluminescence resonance energy transfer, and fluorescence resonance energy transfer (FRET) that at least some GPCRs assemble on the membrane as functional homo-and heterodimers (2-4). Dimerization of GPCRs has been shown to affect a multitude of receptor functions, including ligand binding, signaling, receptor desensitization, and receptor trafficking (2-5).The mechanism by which GPCR dimerization occurs remains obscure and controversial. One model suggests that ligand binding induces a conformational change in the receptor that favors dimer formation. In contrast to this model, the presence of GPCRs, which may be assembled as preformed dimers, has been shown for members of the class C subfamily, which includes GABAergic receptors (6 -8), calcium-sensing receptor (9, 10), the metabotropic glutamate receptor (11), and the sweet taste receptors (12)(13)(14). This paradigm of GPCR assembly, however, is not consistent among the other families of GPCRs (2). Several reports have shown that agonist plays an active role in GPCR dimerization at the plasma membrane, suggesting an equilibrium between GPCR dimers or monomers that can be regulated by ligand occupancy. These receptors include the human somatostatin receptors (hSSTRs) (15, 16), the dopamine D2 receptor (17), the gonadotropin-releasing hormone receptor (18,19), the luteinizing hormone/chorionic gonadotropin hor...

Purpose To establish relationships between quantitative magnetic resonance imaging (qMRI) and biomechanical parameters to help inform and interpret alterations of human intervertebral discs (IVD) with different grades of degeneration. Materials and Methods The properties of the nucleus pulposus (NP) and annulus fibrosus (AF) tissues of each IVD of 10 lumbar spines (range 32–77 years) were analyzed by qMRI (relaxation times T1 and T2, magnetization transfer ratio MTR and apparent diffusion coefficient ADC), and tested in confined compression and dynamic shear. Results T1 and T2 significantly decreased in both the NP and AF with increasing degeneration grades while the MTR increased significantly with grade 4. In contrast with the others qMRI parameters, the ADC had a tendency to decrease with increasing grade. Disc degeneration caused a decrease in the aggregate modulus, hydraulic permeability and shear modulus magnitude along with an increase in phase angle in the AF. On the other hand, disc degeneration of NPs decreased the shear modulus and the phase angle. Conclusion Our studies indicate that qMRI can be used as a non-invasive diagnostic tool in the detection of IVDs properties with potential to help interpret and to detect early, middle and late stages of degeneration. QMRI of the human IVD can therefore become a very important diagnostic assessment tool in determining the functional state of the disc.

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