Retromer terminates the generation of cAMP by internalized PTH receptors.

The generation of cAMP by G protein-coupled receptors (GPCRs) and its termination are currently thought to occur exclusively at the plasma membrane of cells. Under existing models of receptor regulation, this signal is primarily restricted by desensitization of the receptors through their binding to ß-arrestins. However, this paradigm is not consistent with recent observations that the parathyroid hormone receptor type 1 (PTHR) continues to stimulate cAMP production even after receptor internalization, as ß-arrestins are known to rapidly bind and internalize activated PTHR. Here we show that binding to ß-arrestin1 prolongs rather than terminates the generation of cAMP by PTHR, and that cAMP generation correlates with the persistence of arrestin-receptor complexes on endosomes. PTHR signaling is instead turned off by the retromer complex, which regulates the movement of internalized receptor from endosomes to the Golgi apparatus. Thus, binding by the retromer complex regulates the sustained generation of cAMP triggered by an internalized GPCR.

Sustained cyclic AMP production by parathyroid hormone receptor endocytosis.

Cell signaling mediated by the G protein-coupled parathyroid hormone receptor type 1 (PTHR) is fundamental to bone and kidney physiology. It has been unclear how the two ligand systems--PTH, endocrine and homeostatic, and PTH-related peptide (PTHrP), paracrine--can effectively operate with only one receptor and trigger different durations of the cAMP responses. Here we analyze the ligand response by measuring the kinetics of activation and deactivation for each individual reaction step along the PTHR signaling cascade. We found that during the time frame of G protein coupling and cAMP production, PTHrP(1-36) action was restricted to the cell surface, whereas PTH(1-34) had moved to internalized compartments where it remained associated with the PTHR and Galpha(s), potentially as a persistent and active ternary complex. Such marked differences suggest a mechanism by which PTH and PTHrP induce differential responses, and these results indicate that the central tenet that cAMP production originates exclusively at the cell membrane must be revised.

Prolonged signaling at the parathyroid hormone receptor by peptide ligands targeted to a specific receptor conformation.

The parathyroid hormone receptor (PTHR) is a class B G protein-coupled receptor that plays critical roles in bone and mineral ion metabolism. Ligand binding to the PTHR involves interactions to both the amino-terminal extracellular (N) domain, and transmembrane/extracellular loop, or juxtamembrane (J) regions of the receptor. Recently, we found that PTH(1-34), but not PTH-related protein, PTHrP(1-36), or M-PTH(1-14) (M = Ala/Aib(1),Aib(3),Gln(10),Har(11),Ala(12),Trp(14),Arg(19)), binds to the PTHR in a largely GTPgammaS-resistant fashion, suggesting selective binding to a novel, high-affinity conformation (R(0)), distinct from the GTPgammaS-sensitive conformation (RG). We examined the effects in vitro and in vivo of introducing the M substitutions, which enhance interaction to the J domain, into PTH analogs extended C-terminally to incorporate residues involved in the N domain interaction. As compared with PTH(1-34), M-PTH(1-28) and M-PTH(1-34) bound to R(0) with higher affinity, produced more sustained cAMP responses in cells, formed more stable complexes with the PTHR in FRET and subcellular localization assays, and induced more prolonged calcemic and phosphate responses in mice. Moreover, after 2 weeks of daily injection in mice, M-PTH(1-34) induced larger increases in trabecular bone volume and greater increases in cortical bone turnover, than did PTH(1-34). Thus, the putative R(0) PTHR conformation can form highly stable complexes with certain PTH ligand analogs and thereby mediate surprisingly prolonged signaling responses in bone and/or kidney PTH target cells. Controlling, via ligand analog design, the selectivity with which a PTH ligand binds to R(0), versus RG, may be a strategy for optimizing signaling duration time, and hence therapeutic efficacy, of PTHR agonist ligands.

Conformational cross-talk between alpha2A-adrenergic and mu-opioid receptors controls cell signaling.

Morphine, a powerful analgesic, and norepinephrine, the principal neurotransmitter of sympathetic nerves, exert major inhibitory effects on both peripheral and brain neurons by activating distinct cell-surface G protein-coupled receptors-the mu-opioid receptor (MOR) and alpha2A-adrenergic receptor (alpha2A-AR), respectively. These receptors, either singly or as a heterodimer, activate common signal transduction pathways mediated through the inhibitory G proteins (G(i) and G(o)). Using fluorescence resonance energy transfer microscopy, we show that in the heterodimer, the MOR and alpha2A-AR communicate with each other through a cross-conformational switch that permits direct inhibition of one receptor by the other with subsecond kinetics. We discovered that morphine binding to the MOR triggers a conformational change in the norepinephrine-occupied alpha2A-AR that inhibits its signaling to G(i) and the downstream MAP kinase cascade. These data highlight a new mechanism in signal transduction whereby a G protein-coupled receptor heterodimer mediates conformational changes that propagate from one receptor to the other and cause the second receptor's rapid inactivation.

Rationalizing alpha-helical membrane protein crystallization.

X-ray crystallography is currently the most successful method for determining the three-dimensional structure of membrane proteins. Nevertheless, growing the crystals required for this technique presents one of the major bottlenecks in this area of structural biology. This is especially true for the alpha-helical type membrane proteins that are of particular interest due to their medical relevance. To address this problem we have undertaken a detailed analysis of the crystallization conditions from 121 alpha-helical membrane protein structures deposited in the Protein Data Bank. This information has been analyzed so that the success of different parameters can be easily compared for different membrane protein families. Concurrent with this analysis, we also present the new sparse matrix crystallization screen MemGold.

Crystal structures of an intein from the split dnaE gene of Synechocystis sp. PCC6803 reveal the catalytic model without the penultimate histidine and the mechanism of zinc ion inhibition of protein splicing.

The first naturally occurring split intein was found in the dnaE gene of Synechocystis sp. PCC6803 and belongs to a subclass of inteins without a penultimate histidine residue. We describe two high-resolution crystal structures, one derived from an excised Ssp DnaE intein and the second from a splicing-deficient precursor protein. The X-ray structures indicate that His147 in the conserved block F activates the side-chain N(delta) atom of the intein C-terminal Asn159, leading to a nucleophilic attack on the peptide bond carbonyl carbon atom at the C-terminal splice site. In this process, Arg73 appears to stabilize the transition state by interacting with the carbonyl oxygen atom of the scissile bond. Arg73 also seems to substitute for the conserved penultimate histidine residue in the formation of an oxyanion hole, as previously identified in other inteins. The finding that the precursor structure contains a zinc ion chelating the highly conserved Cys160 and Asp140 reveals the structural basis of Zn2+-mediated inhibition of protein splicing. Furthermore, it is of interest to observe that the carbonyl carbon atom of Asn159 and N(eta) of Arg73 are 2.6 angstroms apart in the free intein structure and 10.6 angstroms apart in the precursor structure. The orientation change of the aromatic ring of Tyr-1 following the initial acyl shift may be a key switching event contributing to the alignment of Arg73 and the C-terminal scissile bond, and may explain the sequential reaction property of the Ssp DnaE intein.

A single surface tryptophan in the chitin-binding domain from Bacillus circulans chitinase A1 plays a pivotal role in binding chitin and can be modified to create an elutable affinity tag.

Site-directed mutagenesis was carried out to investigate the roles of a number of highly conserved residues of the chitin-binding domain (ChBD) of Bacillus circulans chitinase A1 (ChiA1) in the binding of chitin. Analysis of single alanine replacement mutants showed that mutation of an exposed tryptophan residue (Trp(687)) impaired the binding to chitin, while mutation of other highly conserved residues, most carrying aromatic or hydrophobic side chains, did not significantly affect the binding activity. Interestingly, replacement of Trp(687) with phenylalanine significantly reduced chitin-binding activity at lower salt concentrations (0-1 M NaCl) but allowed strong binding to chitin at 2 M NaCl. Since Trp(687) is conserved among the ChBDs belonging to the bacterial ChiA1 subfamily, the data presented suggest a general mechanism in which this exposed tryptophan, which is located in the cleft formed between two beta-sheets as revealed by the solution structure [J. Biol. Chem. 275 (2000) 13654], makes a major contribution to ligand binding presumably through hydrophobic interactions. Furthermore, modulation of the chitin-binding activity by the conserved amino acid replacement (W687F) and a shift in the ionic strength of buffer has led to the development of an elutable affinity tag for single column purification of recombinant proteins.

Purification and initial crystallization studies of a DnaB intein from Synechocystis sp. PCC 6803.

A 154-residue mini-intein from the dnaB gene of Synechocystis sp. PCC 6803 (Ssp DnaB intein) has been purified and crystallized using PEG 4000 as a precipitant. The crystal belongs to space group P3(1)21 or P3(2)21, with unit-cell parameters a = b = 58.2, c = 70.3 A. It has one molecule per asymmetric unit and diffracts to beyond 2.0 A under cryoconditions (100 K) using a rotating copper anode X-ray generator.

Crystallographic study of a naturally occurring trans-splicing intein from Synechocystis sp. PCC 6803.

A naturally occurring split intein from the dnaE gene of Synechocystis sp. PCC6803 (Ssp DnaE intein) has been purified and crystallized using PEG 8K as precipitant. The crystal belongs to space group P3(1)21 or P3(2)21, with unit-cell parameters a = b = 58.5, c = 70.2 A. It has one molecule per asymmetric unit and diffracts to beyond 2.9 A under cryoconditions (110 K) using a Cu rotating-anode X-ray generator in-house.

Crystal structure of a mini-intein reveals a conserved catalytic module involved in side chain cyclization of asparagine during protein splicing.

We have determined the crystal structure of a 154-residue intein derived from the dnaB gene of Synechocystis sp. strain PCC6803 and refined it to a 2.0-A resolution. The x-ray structure suggests that this intein possesses two catalytic sites that appear to be separately responsible for splicing and cleavage of the N- and C-terminal scissile bonds. The conserved intein block F residues are the important components of a catalytic site for side chain cyclization of the last intein residue, Asn-154. The data suggest that the imidazole ring of His-143 is involved in the activation of the side chain Ndelta atom of Asn-154, leading to a nucleophilic attack on the carbonyl carbon of Asn-154. Substitution of His-143 with Ala or Gln resulted in the inhibition of C-terminal cleavage. His-153, Asp-136, and a water molecule appear to constitute an oxyanion binding site by contacting the carbonyl oxygen of Asn-154 to stabilize the transition state. The structure and mutagenesis data also support that the close contact between the hydroxyl groups of Thr-138 and Ser-155, whose side chain participates in an S --> O acyl shift, plays an important role in the nucleophile orientation. Our structural modeling suggests that this catalytic module is conserved in the C-terminal subdomains of inteins from diverse organisms.