Kaleeckal G. Harikumar

Dependence of critical micelle concentration of a zwitterionic detergent on ionic strength: implications in receptor solubilization

The zwitterionic detergent, 3‐[(3‐cholamidopropyl)‐dimethylammonio]‐1‐propanesulfonate (CHAPS), is mild, non‐denaturing, and extensively used for solubilizing membrane proteins and receptors. We report here that the critical micelle concentration (CMC) of CHAPS is dependent on the concentration of NaCl in the solution. Thus, the CMC of CHAPS decreases from 6.41 mM in absence of any salt to 4.10 mM in presence of 1.5 M NaCl. The logarithm of the CMC values appear to have a linear relationship with the salt concentration. Such changes in CMC with ionic strength have important implications in solubilization of membrane‐bound neuronal receptors. This is shown by optimal solubilization of the serotonin receptor type 1A (5‐HT1A) from bovine brain hippocampal crude (native) membrane by CHAPS at premicellar concentration under high salt conditions.

Transmembrane segment IV contributes a functionally important interface for oligomerization of the class II G protein-coupled secretin receptor

Oligomerization of the Class II G protein-coupled secretin receptor has been reported, but the molecular basis for this and its functional significance have not been determined. In the current work, we have examined the possible contribution of each of the transmembrane (TM) segments of this receptor to its homo-oligomerization, using the method of competitive disruption screening for inhibition of receptor bioluminescence resonance energy transfer signal. TM IV was the only segment that was found to disrupt receptor bioluminescence resonance energy transfer. Evaluation of predicted interhelical and lipid-exposed faces of this TM segment demonstrated that its lipid-exposed face represented the determinant for oligomerization. This was further confirmed by mutagenesis of the intact secretin receptor. Morphological FRET was utilized to demonstrate that secretin receptor oligomerization occurred at the cell surface and that this oligomerization was disrupted by mutating Gly243 and Ile247, key residues within the lipid-exposed face of TM IV. Although disruption of the receptor oligomerization interface had no effect on secretin binding parameters, it reduced the ability of secretin to stimulate intracellular cAMP. This supports a clear functional effect of oligomerization of this receptor. Such an effect might be particularly relevant to clinical situations in which this receptor is overexpressed, such as in certain neoplasms.

Constitutive Formation of Oligomeric Complexes between Family B G Protein-Coupled Vasoactive Intestinal Polypeptide and Secretin Receptors

Formation of oligomeric complexes of family A G protein-coupled receptors has been shown to influence their function and regulation. However, little is known about the existence of such complexes for family B receptors in this superfamily. We previously used bioluminescence resonance energy transfer (BRET) to demonstrate that the prototypic family B secretin receptor forms ligand-independent oligomeric complexes. Here, we show that subtypes of human vasoactive intestinal polypeptide receptors (VPAC1 and VPAC2) that represent the closest structurally related receptors to the secretin receptor also form constitutive oligomers with themselves and with the secretin receptor. We prepared tagged constructs expressing Renilla reniformis luciferase, yellow fluorescent protein, or cyan fluorescent protein at the carboxyl terminus of VPAC1, VPAC2, and secretin receptors, and performed BRET and morphologic fluorescence resonance energy transfer (FRET) studies with all combinations. The specificity of the BRET and FRET signals was confirmed by control studies. These constructs bound their natural ligands specifically and saturably, with these agonists able to elicit full cAMP responses. BRET studies showed that, like the secretin receptor, both VPAC receptors exhibited constitutive homo-oligomerization in COS cells. Unlike secretin receptor oligomers that were unaffected by ligand binding, the VPAC receptor homo-oligomers were modulated by vasoactive intestinal polypeptide. In addition, each of these three receptors formed hetero-oligomers with each other. The VPAC1-VPAC2 hetero-oligomers were modulated by vasoactive intestinal polypeptide binding, whereas the secretin-VPAC1 and secretin-VPAC2 receptor hetero-oligomers were unaffected by ligand treatment. Morphologic FRET studies demonstrated that each of the homo-oligomers and the VPAC1-VPAC2 receptor hetero-oligomers reached the cell surface, where receptor interactions were clear. However, coexpression of secretin receptors with either type of VPAC receptor resulted in intracellular trapping of the hetero-oligomeric complexes within the biosynthetic pathway. These studies provide new insight into the ability of family B G protein-coupled receptors to associate with each other in cells.

Heterodimerization of Type A and B Cholecystokinin Receptors Enhance Signaling and Promote Cell Growth

Dimerization of several G protein-coupled receptors has recently been described, but little is known about its clinical and functional relevance. Cholecystokinin (CCK) and gastrin are structurally related gastrointestinal and neuronal peptides whose functions are mediated by two structurally related receptors in this superfamily, the type A and B CCK receptors. We previously demonstrated spontaneous homodimerization of type A CCK receptors and the dissociation of those complexes by agonist occupation (Cheng, Z. J., and Miller, L. J. (2001) J. Biol. Chem. 276, 48040-48047). Here, for the first time, we also demonstrate spontaneous homodimerization of type B CCK receptors, as well as heterodimerization of that receptor with the type A CCK receptor. Unlike type A CCK receptor dimers, the homodimerization of type B CCK receptors was not affected by ligand occupation. However, although heterodimers of type A and B CCK receptors bound natural agonists normally, they exhibited unusual functional and regulatory characteristics. Such complexes demonstrated enhanced agonist-stimulated cellular signaling and delayed agonist-induced receptor internalization. As a likely consequence, agonist-stimulated cell growth was markedly enhanced in cells simultaneously expressing both of these receptors. Our results provide the first evidence that heterodimerization of G protein-coupled receptors can form a more “powerful” signaling unit, which has potential clinical significance in promoting cell growth.

Structure and function of Norrin in assembly and activation of a Frizzled 4-Lrp5/6 complex.

Norrin is a cysteine-rich growth factor that is required for angiogenesis in the eye, ear, brain, and female reproductive organs. It functions as an atypical Wnt ligand by specifically binding to the Frizzled 4 (Fz4) receptor. Here we report the crystal structure of Norrin, which reveals a unique dimeric structure with each monomer adopting a conserved cystine knot fold. Functional studies demonstrate that the novel Norrin dimer interface is required for Fz4 activation. Furthermore, we demonstrate that Norrin contains separate binding sites for Fz4 and for the Wnt ligand coreceptor Lrp5 (low-density lipoprotein-related protein 5) or Lrp6. Instead of inducing Fz4 dimerization, Norrin induces the formation of a ternary complex with Fz4 and Lrp5/6 by binding to their respective extracellular domains. These results provide crucial insights into the assembly and activation of the Norrin-Fz4-Lrp5/6 signaling complex.

Dimeric arrangement of the parathyroid hormone receptor and a structural mechanism for ligand-induced dissociation

The parathyroid hormone receptor (PTH1R) is a class B G protein-coupled receptor that is activated by parathyroid hormone (PTH) and PTH-related protein (PTHrP). Little is known about the oligomeric state of the receptor and its regulation by hormone. The crystal structure of the ligand-free PTH1R extracellular domain (ECD) reveals an unexpected dimer in which the C-terminal segment of both ECD protomers forms an α-helix that mimics PTH/PTHrP by occupying the peptide binding groove of the opposing protomer. ECD-mediated oligomerization of intact PTH1R was confirmed in living cells by bioluminescence and fluorescence resonance energy transfer experiments. As predicted by the structure, PTH binding disrupted receptor oligomerization. A receptor rendered monomeric by mutations in the ECD retained wild-type PTH binding and cAMP signaling ability. Our results are consistent with the hypothesis that PTH1R forms constitutive dimers that are dissociated by ligand binding and that monomeric PTH1R is capable of activating G protein.

Differential discrimination of G-protein coupling of serotonin1A receptors from bovine hippocampus by an agonist and an antagonist

We have studied the effect of guanosine‐5′‐O‐(3‐thiotriphosphate) (GTP‐γ‐S), a non‐hydrolyzable analogue of GTP, on agonist and antagonist binding to bovine hippocampal 5‐hydroxytryptamine (5‐HT)1A receptor in native membranes. Our results show that the specific binding of the agonist is inhibited with increasing concentrations of GTP‐γ‐S along with a reduction in binding affinity. In sharp contrast to this, antagonist binding to 5‐HT1A receptor shows no significant reduction and remains invariant over a large range of GTP‐γ‐S concentrations. The binding affinity of the antagonist also remains unaltered. This shows that the agonist and the antagonist differentially discriminate G‐protein coupling of 5‐HT1A receptors from bovine hippocampus.

Solubilization of high affinity G-protein-coupled serotonin 1A receptors from bovine hippocampus using pre-micellar CHAPS at low concentration

The serotonin 1A (5-HT 1A ) receptors are members of a superfamily of seven transmembrane domain receptors that couple to G-proteins. They appear to be involved in various behavioural and cognitive functions. This paper reports an efficient strategy to solubilize 5-HT 1A receptors from bovine hippocampal membranes using the zwitterionic detergent CHAPS which is mild and non-denaturing. Since high concentration of CHAPS has earlier been shown to induce dissociation and depletion of G-protein sub-units, a low (pre-micellar) concentration of CHAPS was used for solubilizing 5-HT 1A receptors in the presence of NaCl followed by PEG precipitation. This results in solubilization of 5-HT 1A receptors with a high degree of efficiency and gives rise to high affinity, functionally active G-protein-sensitive solubilized receptors. Optimal solubilization of the receptor from the native source with high ligand binding affinity and intact signal transduction components may constitute the first step in the molecular characterization of the 5-HT 1A receptor in particular, and G-protein-coupled receptors in general.

Pattern of intra-family hetero-oligomerization involving the G-protein-coupled secretin receptor.

Oligomerization of G-protein-coupled receptors (GPCRs) is emerging as a mechanism for regulation and functional modification, although it has been studied most extensively for Family A receptors. Family B receptors have clear structural differences from Family A. In this paper, we have systematically evaluated GPCRs that are capable of association with the prototypic Family B secretin receptor. All of the receptor constructs were shown to traffic normally to the plasma membrane. We utilized receptor bioluminescence resonance energy transfer (BRET) to determine the presence of constitutive and ligand-dependent receptor association. Extensive intra-family and no cross-family association was observed. Of the nine Family B receptors studied, all constitutively yielded a significant BRET signal with the secretin receptor, except for the calcitonin receptor. Each of the associating hetero-oligomeric receptor pairs generated a BRET signal of similar intensity, less than that of homo-oligomeric secretin receptors. BRET signals from some receptor pairs were reduced by ligand occupation, but none were increased by this treatment. Thus, Family B GPCR oligomerization occurs, with many structurally related members associating with each other. The specific functional implications of this need to be further evaluated.

Dimerization in the Absence of Higher Order Oligomerization of the G Protein-Coupled Secretin Receptor

Oligomerization of G protein-coupled receptors has been proposed to affect receptor function and regulation; however, little is known about the molecular nature of such complexes. We previously utilized bioluminescence resonance energy transfer (BRET) to demonstrate that the prototypic Family B secretin receptor can form oligomers. We now explore the order of oligomerization present utilizing unique bimolecular fluorescence complementation and energy transfer techniques. The non-fluorescent carboxyl-terminal and amino-terminal halves of yellow fluorescent protein (YFP) were fused to the carboxyl terminus of the secretin receptor. These constructs bound secretin normally and signaled in response to secretin like wild type receptor. When co-expressed on COS cells, these constructs physically interacted to yield typical YFP fluorescence in biosynthetic compartments and at the plasma membrane, reflecting receptor homo-dimerization. However, the addition of another potential partner in form of Rlu- or CFP-tagged secretin receptor yielded no significant BRET or FRET signal, respectively, under conditions in which intact YFP-tagged secretin receptor yielded such a signal. Absence of higher-order receptor oligomers was further confirmed using saturation BRET techniques. Absence of significant resonance transfer to the secretin receptor homo-dimer was true for carboxyl-terminally-tagged secretin receptor, as well as for receptor incorporating the transfer partner into each of the three distinct intracellular loop domains. These results suggest that the secretin receptor can exist only as a structurally-specific homo-dimer, without being present as higher-order oligomers.