Enhanced hypotensive, bradycardic, and hypnotic responses to alpha2-adrenergic agonists in spinophilin-null mice are accompanied by increased G protein coupling to the alpha2A-adrenergic receptor.

We previously identified spinophilin as a regulator of alpha(2) adrenergic receptor (alpha(2)AR) trafficking and signaling in vitro and in vivo (Science 304:1940-1944, 2004). To assess the generalized role of spinophilin in regulating alpha(2)AR functions in vivo, the present study examined the impact of eliminating spinophilin on alpha(2)AR-evoked cardiovascular and hypnotic responses, previously demonstrated to be mediated by the alpha(2A)AR subtype, after systemic administration of the alpha(2)-agonists 5-bromo-N-(4,5-dihydro-1H-imidazol-2-yl)-6-quinoxalinamine (UK14,304) and clonidine in spinophilin-null mice. Mice lacking spinophilin expression display dramatically enhanced and prolonged hypotensive, bradycardic, and sedative-hypnotic responses to alpha(2)AR stimulation. Whereas these changes in sensitivity to alpha(2)AR agonists occur independent of any changes in alpha(2A)AR density or intrinsic affinity for agonist in the brains of spinophilin-null mice compared with wild-type control mice, the coupling of the alpha(2A)AR to cognate G proteins is enhanced in spinophilin-null mice. Thus, brain preparations from spinophilin-null mice demonstrate enhanced guanine nucleotide regulation of UK14,304 binding and evidence of a larger fraction of alpha(2A)AR in the guanine-nucleotide-sensitive higher affinity state compared with those from wild-type mice. These findings suggest that eliminating spinophilin expression in native tissues leads to an enhanced receptor/G protein coupling efficiency that contributes to sensitization of receptor mediated responses in vivo.

A point mutation of the alpha 2-adrenoceptor that blocks coupling to potassium but not calcium currents.

The alpha 2A-adrenergic receptor (adrenoceptor) was stably expressed in AtT20 mouse pituitary tumor cells; adrenoceptor agonists inhibited adenylyl cyclase, inhibited voltage-dependent calcium currents, and increased inwardly rectifying potassium currents. An aspartic acid residue (Asp79) highly conserved among guanine nucleotide-binding protein (G protein)-coupled receptors was mutated to asparagine; in cells transfected with the mutant alpha 2-receptor, agonists inhibited adenylyl cyclase and calcium currents but did not increase potassium currents. Because distinct G proteins appear to couple adrenoceptors to potassium and calcium currents, the present findings suggest that the mutant alpha 2-adrenoceptor cannot achieve the conformation necessary to activate G proteins that mediate potassium channel activation.

Central hypotensive effects of the alpha2a-adrenergic receptor subtype.

alpha2-Adrenergic receptors (alpha2ARs) present in the brainstem decrease blood pressure and are targets for clinically effective antihypertensive drugs. The existence of three alpha2AR subtypes, the lack of subtype-specific ligands, and the cross-reactivity of alpha2AR agonists with imidazoline receptors has precluded an understanding of the role of individual alpha2AR subtypes in the hypotensive response. Gene targeting was used to introduce a point mutation into the alpha2aAR subtype in the mouse genome. The hypotensive response to alpha2AR agonists was lost in the mutant mice, demonstrating that the alpha2aAR subtype plays a principal role in this response.

Leukocyte beta-receptor alterations in hypertensive subjects.

It has been suggested that beta-adrenergic responsiveness is reduced in hypertension. To evaluate a possible alteration in human beta-receptors that might account for diminished beta-adrenergic responsiveness, we studied leukocytes from hypertensive and normotensive subjects after an overnight rest supine, and then after being ambulatory, a maneuver that increases plasma catecholamines approximately twofold. In supine samples, beta-receptor affinity for the agonist isoproterenol was significantly reduced in hypertensives and was associated with a reduction in the proportion of beta-receptors binding agonist with a high affinity from 42 +/- 6% in normotensive subjects to 25 +/- 2% in hypertensives (P less than 0.05). Alterations in beta-adrenergic-mediated adenylate cyclase activity parallelled the differences seen in the beta-receptor affinity for agonist. In normotensive subjects, beta-receptor density and the proportion of receptors binding agonist with high affinity were reciprocally correlated with plasma catecholamines. However, in the hypertensive subjects these correlations were not evident. Thus, our data suggest an alteration in leukocyte beta-receptor interactions in hypertensive subjects, and may represent a generalized defect in beta-receptor function in hypertension.

Dynamic regulation of leukocyte beta adrenergic receptor-agonist interactions by physiological changes in circulating catecholamines.

beta-Adrenergic receptors on human mononuclear leukocytes were assessed using [125I]iodohydroxybenzylpindolol binding. Subjects were studied supine and after being ambulatory, a maneuver that increases plasma catecholamines approximately two-fold. beta-Receptor affinity for agonists, measured by the competition of [125I]iodohydroxybenzylpindolol binding by (-)isoproterenol was significantly reduced with ambulation and this reduction was associated with a reduction in the proportion of beta-receptors binding agonist with a high affinity from a mean (+/- SEM) of 42 +/- 5 to 24 +/- 2% (P less than 0.01). In a parallel series, beta-adrenergic-stimulated adenylate cyclase activity was also reduced with postural change from 4.6 +/- 1.1 to 2.4 +/- 0.6 pmol [32P]cAMP/min per mg protein (P less than 0.05) after ambulation. Similar reductions in the proportion of receptors binding agonist with a high affinity were seen after infusion of norepinephrine. We conclude that the maneuver of ambulation reduces leukocyte beta-receptor responsiveness and affinity for agonists, probably by the effect of increased plasma catecholamines mediating an uncoupling of the beta-receptor-adenylate cyclase complex.

The alpha(2a)-adrenergic receptor plays a protective role in mouse behavioral models of depression and anxiety.

The noradrenergic system is involved in the regulation of many physiological and psychological processes, including the modulation of mood. The alpha(2)-adrenergic receptors (alpha(2)-ARs) modulate norepinephrine release, as well as the release of serotonin and other neurotransmitters, and are therefore potential targets for antidepressant and anxiolytic drug development. The current studies were undertaken to examine the role of the alpha(2A) subtype of alpha(2)-AR in mouse behavioral models of depression and anxiety. We have observed that the genetic knock-out of the alpha(2A)-AR makes mice less active in a modified version of Porsolt's forced swim test and insensitive to the antidepressant effects of the tricyclic drug imipramine in this paradigm. Furthermore, alpha(2A)-AR knock-out mice appear more anxious than wild-type C57 Bl/6 mice in the rearing and light-dark models of anxiety after injection stress. These findings suggest that the alpha(2A)-AR may play a protective role in some forms of depression and anxiety and that the antidepressant effects of imipramine may be mediated by the alpha(2A)-AR.

Myocardial guanylate cyclase: properties of the enzyme and effects of cholinergic agonists in vitro.

The characteristics of myocardial guanylate cyclase (GTP pyrophosphatelyase, EC 4.6.1.2) were studied. Specific activity of the myocardial enzyme in five vertebrate species was guinea pig greater than man greater than cat greater than dog greater than rat. In the guinea pig, guanylate cyclase activity was uniformly distributed throughout the anatomical regions of the heart. The major portion of the enzyme activity was retrieved in the supernatant fraction after centrifugation at 12 000 times g. The Km for GTP was similar in supernatant (0.12 mM) and particulate (0.21 mM) preparations, although the Ka for Mn2+ in particulate preparations (0.3-0.6 mM) was less than that observed for guanylate cyclase in the supernatant fraction (0.8-2.0 mM). ATP competitively inhibited supernatant and particulate activity. Addition of 0.005-10.0 mM Ca2+ to assay incubations did not enhance guanylate cyclase activity. Suspension of 105 000 times g supernatant guanylate cyclase preparations with membrane lipids or phosphatidylserine stimulated activity 1.4-4.3 fold, whereas similar treatment of particulate preparations caused little alteration of enzyme activity. Addition of the cholinergic agonists acetylcholine, carbachol or methacholine (10-4-10-8 M) to homogenate, supernatant, particulate and disrupted tissue slice preparations in the presence of 0.0012-1.2 mM GTP, 0.3-10.0 mM Mn2+ and 0.005-10.0 mM Ca2+ or 0.0012-1.2 mM ATP did not stimulate guanylate cyclase activity. Similarly, further stimulation of guanylate cyclase activity was not elicited when enzyme-lipid suspensions were assayed in the presence of cholinergic agents.

Human platelet phospholipase A2 activity is responsive in vitro to pH and Ca2+ variations which parallel those occurring after platelet activation in vivo.

Secretion of human platelet dense granule contents in response to epinephrine and other weak agonists requires the prior liberation of membrane-esterified arachidonic acid by a phospholipase A2 enzyme species whose activity is regulated by Na+/H+ exchange (e.g., Sweatt et al. (1986) J. Biol. Chem. 261, 8660-8673 and Banga et al. (1986) Proc. Natl. Acad. Sci. USA 83, (197-9201). Based on our earlier findings in intact platelets, we postulated that the alkalinization of the platelet interior that accompanies accelerated activity of the Na+/H+ antiporter enables the phospholipase A2 enzyme to function at ambient or low concentrations of intraplatelet Ca2+. To test the hypothesis that the Ca2+ dependence of platelet phospholipase A2 activity is influenced by changes in intraplatelet pH that occur following platelet activation, we characterized the Ca2+ dependence of this enzyme as a function of changes in pH (from pH 6.8-8.0), since it is within this range that intraplatelet pH changes occur following platelet activation. Phospholipase A2 enzymatic activity in platelet particulate preparations was detectable in the presence of micromolar concentrations of Ca2+ (EC50 1-2 microM) and plateaued above 10 microM Ca2+. Enzymatic activity measured at 4.8 microM Ca2+ was increased by raising the pH from 5.5 to 8.0 (EC50 7.4), was optimal at pH 8.0 and declined at more alkaline values. Furthermore, increases in pH from pH 6.8 to pH 8.0 not only increased maximal enzymatic activity but also enabled detection of enzymatic activity at lower Ca2+ concentrations. The interdependent regulation of phospholipase A2 activity by changes in pH and Ca2+ suggests that phospholipase A2 could serve to integrate changes in intracellular pH and available Ca2+ that occur subsequent to activation of human platelets by epinephrine and other weak agonists.

Mn2+-uncoupling of the catecholamine-sensitive adenylate cyclase system of rat reticulocytes. Parallel effects on cholera toxin-catalyzed ADP-ribosylation of the system.

High concentrations of Mn2+ interfere with functional interactions between the GTP-binding regulatory protein (G) and the catalytic moiety (C) of adenylate cyclase without perturbing interactions between receptor (R) and component G in rat reticulocyte membranes. The ability of cholera toxin to ADP-ribosylate component G and to enhance GTP-stimulated adenylate cyclase activity also appears to be correlated with the efficacy of the communication of component G with the adenylate cyclase system. Thus, increasing the concentration of Mn2+ in rat reticulocyte membrane during in vitro incubations causes a parallel loss of Gpp(NH)p-stimulated adenylate cyclase activity, cholera toxin-catalyzed [32P]ADP-ribosylation of the 42 000 Mr subunit of component G and cholera toxin-catalyzed enhancement of GTP-sensitive adenylate cyclase activity. Removal of Mn2+ by washing the membranes completely restores the sensitivity of adenylate cyclase activity. Removal of Mn2+ by washing the membranes completely restores the sensitivity of adenylate cyclase to all effectors, including cholera toxin. The data suggest that exposure of membranes to Mn2+ provides a useful tool for reversibly uncoupling catecholamine-sensitive adenylate cyclase systems. The data also suggest that the extent of cholera toxin-catalyzed ADP.-ribosylation of membrane substrates, i.e., the G component may rely on functional communication among the various components of the adenylate cyclase system. A corollary of the latter is that the amount of [32P]ADP-ribose-product detected in a membrane may reflect both the quantity and coupling efficiency of component G.

A model system for the biochemical study of luteinizing hormone/chorionic gonadotropin receptor synthesis.

A model system for the biochemical study of LH/CG receptor synthesis has been developed. Culture conditions for porcine granulosa cells were adapted that maximized the selective induction of LH/CG receptors by cAMP-inducing stimuli with an elimination of background LH/CG receptor appearance. It was found that the addition of FSH (1.5 micrograms/ml) or cholera toxin (10 ng/ml) 1 day after plating resulted in optimal induction of the LH/CG receptor (20-60 pg [125I]CG bound/micrograms DNA 72 h after addition) with virtually no LH/CG receptor appearance in the absence of added stimuli. Later additions of FSH or cholera toxin required insulin (1.0 microgram/ml) which alone caused background LH/CG receptor appearance in the absence of any additional stimuli. Furthermore, insulin increased the general rate of cellular protein synthesis, whereas FSH or cholera toxin each decreased it. Thus, the use of FSH or cholera toxin, without insulin, may enable one to detect the synthesis of the LH/CG receptor by metabolic labeling techniques where background protein synthesis has been lowered.

Differential effects of GTP on the coupling of beta-adrenergic receptors to adenylate cyclase from frog and turkey erythrocytes. Application of new methods for the analysis of receptor-effector coupling.

A detailed comparison of the interaction of beta-adrenergic receptors with adenylate cyclase stimulation and modification of this interaction by guanine nucleotides has been made in two model systems, the frog and turkey erythrocyte. Objective analysis of the data was facilitated by the development of new graphical methods which involve the use of logit-logit transformations of percent receptor occupancy versus percent enzyme stimulation plots (coupling curves). Receptor-cyclase coupling in turkey erythrocyte membranes demonstrates a proportional relationship between receptor occupancy and adenylate cyclase activation and is unaffected by exogenous guanine nucleotides. By comparison, the proportional relationship of receptor occupancy and adenylate cyclase activation observed in frog erythrocyte membranes in the absence of guanine nucleotides is modified by the addition of exogenous guanine nucleotides such that a greater fractional enzyme stimulation is elicited by low receptor occupancy. Methodological criteria crucial for valid comparison of receptor occupancy and adenylate cyclase activity are delineated. In addition, the possible molecular mechanisms of receptor-cyclase coupling which might give rise to the coupling curves observed are discussed.

Localization of G-protein-coupled receptors in health and disease.

G-protein-coupled receptors (GPCRs) represent a superfamily of proteins, characterized by seven transmembrane alpha-helices, that signal through interactions with a family of heterotrimeric GTP-binding proteins, referred to as G proteins. The broad range of physiological functions associated with GPCRs indicates that a better understanding of these receptors and their regulation can provide a solid foundation for novel pharmacological interventions in a variety of disease states.

Localization and trafficking of alpha2-adrenergic receptor subtypes in cells and tissues.

The three alpha2-adrenergic receptor (alpha2AR) subtypes, all of which couple to multiple effectors via Gi/Go proteins, perform various functions, including the mediation of decreases in adenylyl cyclase activity, activation of receptor-mediated K+ channels, and inhibition of voltage-gated Ca2+ channels. The alpha2ARs are polarized in many target cells, such as neurons in the peripheral and central nervous system and in intestinal and renal epithelia. Precise targeting and polarization of molecules are crucial for many physiological processes, and may confer a degree of specificity that, in the case of the adrenergic receptors, could represent a reasonable strategy by which catecholamines coordinate cellular function in a highly specific way. Receptors also redistribute in response to agonist occupancy by means of sequestration, endocytosis, recycling, or, alternatively, down-regulation (degradation). The focus of this review is to compare the similarities and differences among the three alpha2AR subtypes in terms of specificity, signaling, and trafficking. It is anticipated that a molecular understanding of receptor trafficking will lead to novel therapeutic strategies for diseases linked to aberrant adrenergic receptor function or localization.

The cAMP-Dependent induction of LH receptors in primary cultures of porcine granulosa cells is not due to the expression of an intracellular pool of LH receptors.

The present studies examined whether the increase in cell surface LH receptors in primary cultures of porcine granulosa cells after exposure to FSH or cholera toxin, agents that increase intracellular cAMP, is due to de novo synthesis of the receptor or to a cAMP-dependent translocation of an intracellular pool of LH receptors to the cell surface. LH receptor induction by FSH was fully inhibited by the addition of cycloheximide to the incubation media, but resumed after cycloheximide was removed. These data suggest that FSH-induced LH receptor appearance requires protein synthesis. However, to be confident that the inhibition of LH receptor appearance did not result from lack of transit of preformed receptors requiring a rapidly turning over pool of proteins, we assayed for possible latent receptors in the cell interior by extracting granulosa cells with Triton X-100. Under conditions which detected about 74% of LH receptors in cells exposed to cholera toxin, little [125]iodo-hCG-binding activity was detected in cells not exposed to a cAMP-promoting stimulus. These findings suggest that a preformed pool of LH receptors does not exist in untreated cells, and that the cAMP-mediated induction of LH receptors requires de novo synthesis of the receptor.

Synthesis and characterization of arylamine derivatives of rauwolscine as molecular probes for alpha 2-adrenergic receptors.

The selective alpha 2-adrenergic receptor antagonist rauwolscine was structurally modified to yield a series of arylamine carboxamide derivatives, which were investigated as potential molecular probes for the localization and structural characterization of alpha 2-adrenergic receptors. The arylamine carboxamides differ in the number of carbon atoms separating the reactive phenyl moiety from the fused ring structure of the parent compound, rauwolscine carboxylate. Competitive inhibition studies with [3H]rauwolscine in rat kidney membranes indicate that the affinity for the carboxamide derivatives is inversely related to the length of the carbon spacer arm with rauwolscine 4-aminophenyl carboxamide (zero carbon spacer arm; rau-AMPC) exhibiting the highest affinity (Kd = 2.3 +/- 0.2 nM). Radioiodination of rau-AMPC yields a ligand, 125I-rau-AMPC, which binds to rat kidney alpha 2-adrenergic receptors with high affinity, as determined by both kinetic analysis (Kd = k2/k1 = 0.016 min-1/2.1 X 10(7) M-1 min-1 = 0.76 nM) and equilibrium binding studies (Kd = 0.78 +/- 0.16 nM). 125I-rau-AMPC was quantitatively converted to the photolabile arylazide derivative 17 alpha-hydroxy-20 alpha-yohimban-16 beta-(N-4-azido-3-[125I]iodophenyl) carboxamide (125I-rau-AZPC). In a partially purified receptor preparation from porcine brain, this compound photolabels a major (Mr = 62,000) peptide. The labeling of this peptide is inhibited by adrenergic agonists and antagonists with a rank order of potency consistent with an alpha 2-adrenergic receptor binding site. Both 125I-rau-AMPC and the photolabile arylazide derivative, 125I-rau-AZPC, should prove useful as molecular probes for the structural and biochemical characterization of alpha 2-adrenergic receptors.

Evaluation of the alpha(2A)-adrenergic receptor gene in a heritable form of temporal lobe epilepsy.

An autosomal dominant form of human temporal lobe epilepsy (TLE) has been mapped to a region of chromosome 10q that contains the intronless alpha(2A)-adrenergic receptor (alpha(2A)AR) gene. Because mutation of the alpha(2A)AR gene in the mouse fosters epileptogenesis, we developed methods for analysis of the alpha(2A)AR coding region applicable to any pathophysiologic state in which the alpha(2A)AR could be implicated in the disease mechanism. This study rules out mutations in the alpha(2A)AR coding region as causal for this form of autosomal dominant TLE.

Synthesis of a yohimbine-agarose matrix useful for large-scale and micropurification of multiple alpha 2-receptor subtypes.

We have provided a detailed protocol for the synthesis of a yohimbine-agarose matrix that has been shown to be effective for isolation of the alpha 2A-adrenergic receptor from human platelet and purification of the alpha 2A-adrenergic receptor to apparent homogeneity from porcine brain cortex using chromatography on only two sequential yohimbine-agarose columns. In addition, this affinity matrix also interacts with alpha 2 receptors of the alpha 2B subtype extracted from cultured NG108-15 cells. Finally, this affinity matrix has proven useful for monitoring posttranslational modifications of the receptor in digitonin extracts of metabolically labeled cells. Thus, this affinity matrix can be exploited for the purification of multiple alpha 2-adrenergic receptor subtypes on both a macro- and microscale and should be of value to any laboratory exploring the molecular basis for alpha 2-adrenergic functions.

Expression of alpha 2-adrenergic receptor subtypes in the mouse brain: evaluation of spatial and temporal information imparted by 3 kb of 5' regulatory sequence for the alpha 2A AR-receptor gene in transgenic animals.

The present studies characterize the expression of the alpha 2A, alpha 2B and alpha 2C adrenergic receptor subtypes via in situ hybridization analysis of messenger RNA expression in the adult mouse brain, as well as the pattern of expression of alpha 2A adrenergic receptor messenger RNA at embryonic day E9.5, the earliest time for detection of the messenger RNA encoding this receptor subtype. alpha 2A adrenergic receptor messenger RNA is highly expressed in the sixth layer of the cortex and the locus coeruleus, alpha 2B adrenergic receptor messenger RNA predominantly in the thalamus and in the Purkinje layer of the cerebellum, and alpha 2C adrenergic receptor messenger RNA in the putamen caudate region of the mouse brain. Both alpha 2A and alpha 2C adrenergic receptor messenger RNA demonstrate strong expression in the amygdaloid complex, hypothalamus, olfactory system and the hippocampal formation. To develop a molecular understanding of the unique cellular expression of messenger RNA encoding the alpha 2A adrenergic receptor subtype, 2.83 kb of the upstream regulatory sequence for the alpha 2A adrenergic receptor gene was fused to the LacZ gene as a reporter gene and expression of beta-galactosidase activity was assessed in transgenic offspring. Although the spatial expression of the transgene in the adult brain often overlaps that for the endogenous alpha 2A adrenergic receptor, both ectopic expression and the absence of appropriate expression were noted; in contrast five of the six lines show temporal expression characteristic of the endogenous alpha 2A adrenergic receptor gene. The present studies provide the first characterization of messenger RNA localization for the three alpha 2 adrenergic receptor subtypes in the mouse CNS. Because the functional roles of the prazosin-sensitive alpha 2B adrenergic receptor and alpha 2C adrenergic receptor subtypes have been masked in most earlier physiological and pharmacological analyses of alpha 2 adrenergic receptor function, identifying the multiple loci alpha 2 adrenergic receptor subtype expression is an important prelude to understanding the functional roles of these three independent receptor populations in the mouse CNS. The findings in the transgenic animals indicating that approximately 3 kb of regulatory sequence has imparted faithful temporal but not spatial expression for the alpha 2A adrenergic receptor gene suggest that additional regulatory information is necessary for appropriate cell specific expression of messenger RNA for the alpha 2A adrenergic receptor subtype.

Gene substitution/knockout to delineate the role of alpha 2-adrenoceptor subtypes in mediating central effects of catecholamines and imidazolines.

Adrenergic receptors form the interface between the sympathetic nervous system and the cardiovascular system as well as many endocrine and parenchymal tissues. For the three alpha 2-adrenergic receptors (alpha 2A, alpha 2B, and alpha 2C), genetic mouse models have been developed that can be used to elucidate the physiologic function of each receptor subtype in vivo. Different strategies for homologous recombination in embryonic stem cells were applied to generate lines of mice with gene knockouts of the individual alpha 2-receptor subtypes (alpha 2A-KO, alpha 2B-KO, and alpha 2C-KO) or with a substitution of a mutant receptor at the wild-type locus (alpha 2-D79N). In these transgenic mice, the cardiovascular effects of alpha 2-agonists and imidazoline receptor agonists were tested. Stimulation of alpha 2B receptors in vascular smooth muscle produces hypertension and counteracts the clinically beneficial hypotensive effect of stimulating alpha 2A receptors in the central nervous system.

Trafficking itineraries of G protein-coupled receptors in epithelial cells do not predict receptor localization in neurons.

These studies explored whether the localization and differential trafficking itineraries of G protein-coupled receptors in polarized renal epithelial cells might predict their localization in neurons, as suggested previously. The A1 adenosine receptor is preferentially localized apically, whereas the three alpha 2-adrenergic receptor (alpha 2 AR) subtypes are localized on the lateral subdomain of Madin-Darby canine kidney cells. The alpha 2A AR and alpha 2C AR subtypes achieve this localization by direct targeting; alpha 2B AR is randomly delivered but preferentially retained basolaterally. Despite their differing itineraries in renal epithelial cells, all three epitope-tagged alpha 2-adrenergic receptor subtypes were found in neuronal cell bodies and along the entire length of the neuronal processes following transfection into long term primary cultures of mouse embryonic spinal cord neurons. In a small fraction of neuronal cells, expression of A1 adenosine receptor was limited to a short segment of their processes, otherwise it too was distributed in the soma and neuronal processes. A mutant alpha 2A AR that exhibits an accelerated turnover on the surface of epithelial cells nonetheless has a localization pattern indistinguishable from the wild-type alpha 2A AR in spinal cord neurons. Thus, unlike examples for GPI-anchored apical proteins that distribute along axons or single transmembrane-spanning basolateral proteins that localize to the soma of neurons, similar predictions do not appear to apply for polytopic G protein-coupled receptors.