The GRK4 subfamily of G protein-coupled receptor kinases. Alternative splicing, gene organization, and sequence conservation.

G protein-coupled receptor kinases (GRKs) desensitize G protein-coupled receptors by phosphorylating activated receptors. The six known GRKs have been classified into three subfamilies based on sequence and functional similarities. Examination of the mouse GRK4 subfamily (GRKs 4, 5, and 6) suggests that mouse GRK4 is not alternatively spliced in a manner analogous to human or rat GRK4, whereas GRK6 undergoes extensive alternative splicing to generate three variants with distinct carboxyl termini. Characterization of the mouse GRK 5 and 6 genes reveals that all members of the GRK4 subfamily share an identical gene structure, in which 15 introns interrupt the coding sequence at equivalent positions in all three genes. Surprisingly, none of the three GRK subgroups (GRK1, GRK2/3, and GRK4/5/6) shares even a single intron in common, indicating that these three subfamilies are distinct gene lineages that have been maintained since their divergence over 1 billion years ago. Comparison of the amino acid sequences of GRKs from various mammalian species indicates that GRK2, GRK5, and GRK6 exhibit a remarkably high degree of sequence conservation, whereas GRK1 and particularly GRK4 have accumulated amino acid changes at extremely rapid rates over the past 100 million years. The divergence of individual GRKs at vastly different rates reveals that strikingly different evolutionary pressures apply to the function of the individual GRKs.

Muscarinic supersensitivity and impaired receptor desensitization in G protein-coupled receptor kinase 5-deficient mice.

G protein-coupled receptor kinase 5 (GRK5) is a member of a family of enzymes that phosphorylate activated G protein-coupled receptors (GPCR). To address the physiological importance of GRK5-mediated regulation of GPCRs, mice bearing targeted deletion of the GRK5 gene (GRK5-KO) were generated. GRK5-KO mice exhibited mild spontaneous hypothermia as well as pronounced behavioral supersensitivity upon challenge with the nonselective muscarinic agonist oxotremorine. Classical cholinergic responses such as hypothermia, hypoactivity, tremor, and salivation were enhanced in GRK5-KO animals. The antinociceptive effect of oxotremorine was also potentiated and prolonged. Muscarinic receptors in brains from GRK5-KO mice resisted oxotremorine-induced desensitization, as assessed by oxotremorine-stimulated [5S]GTPgammaS binding. These data demonstrate that elimination of GRK5 results in cholinergic supersensitivity and impaired muscarinic receptor desensitization and suggest that a deficit of GPCR desensitization may be an underlying cause of behavioral supersensitivity.

Palmitoylation increases the kinase activity of the G protein-coupled receptor kinase, GRK6.

The G protein-coupled receptor kinase GRK6 undergoes posttranslational modification by palmitoylation. Palmitoylated GRK6 is associated with the membrane, while nonpalmitoylated GRK6 remains cytosolic. We have separated palmitoylated from nonpalmitoylated GRK6 to assess their relative kinase activity. Palmitoylated GRK6 is 10-fold more active at phosphorylating beta2-adrenergic receptor than nonpalmitoylated wild-type GRK6 or a nonpalmitoylatable mutant GRK6. A nonpalmitoylatable mutant GRK6 which has been further mutated to undergo posttranslational geranylgeranylation is also more active, recovering most of the activity of the palmitoylated enzyme. This activity increase by lipid modification is expected, as the lipid helps GRK6 localize to cellular membranes where its receptor substrates are found. However, when assayed using a soluble protein (casein) as a substrate, both palmitoylated and prenylated GRK6 display significantly higher activity than nonpalmitoylated wild-type or nonpalmitoylatable mutant GRK6 kinases. This increased activity is not altered by addition of exogenous palmitate or phosphatidycholine vesicles, arguing that it is not due to direct activation of GRK6 by binding palmitate, nor to nonspecific association of the GRK6 with casein. Further, chemical depalmitoylation reduces the casein phosphorylation activity of the palmitoylated, but not prenylated, GRK6 kinase. Thus, palmitoylation of GRK6 appears to play a dual role in increasing the activity of GRK6: it increases the hydrophobicity and membrane association of the GRK6 protein, which helps bring the GRK6 to its membrane-bound substrates, and it increases the kinase catalytic activity of GRK6.

Cloning, characterization, and chromosomal localization of rec1.3, a member of the G-protein-coupled receptor family highly expressed in brain.

During a project to identify G-protein-coupled receptors (GPCR) expressed within taste buds, we have isolated a novel receptor-like sequence. The full length sequence of this receptor (rec1.3) has been obtained in both cow and mouse. Rec1.3 bears little sequence similarity to any GPCR whose ligand is known: the closest identity (33%) is to the orphan receptor edg-1. In cow, rec1.3 is expressed most prominently in the brain, with moderate expression in testis and tongue; in the mouse the expression is more widespread. No specific binding for a range of ligands was detected when the mouse coding sequence was expressed in eukaryotic cells. In situ hybridization showed that rec1.3 is widely expressed throughout the mouse brain and is highly expressed in localized regions of the hindbrain, midbrain and hypothalamus. The rec1.3 gene was localized to the centromeric region of chromosome 4 in mouse, a region associated with neonatal seizures.

Molecular cloning of two cannabinoid type 1-like receptor genes from the puffer fish Fugu rubripes.

The puffer fish, Fugu rubripes (Fugu), has been proposed as a model vertebrate genome. We have characterized two putative G-protein-coupled receptor encoding genes, FCB1A and FCB1B, obtained by degenerate PCR and low-stringency hybridization of a Fugu genomic library. These two genes show high homology to the human cannabinoid receptor type 1 (HCB1), but very low homology to the type 2 receptor. The amino acid sequences of the FCB1A and FCB1B genes are 66.2% identical, and the homology of each gene to HCB1 is 72.2 and 59.0%, respectively. The transcripts of both the FCB1A and the FCB1B receptors are abundant in the brain. No type 2 receptor could be cloned from Fugu. These data suggest that although two cannabinoid receptor-like genes are found in the puffer fish, both show similarity to the type 1 receptor found in human.

Characterization of the G protein-coupled receptor kinase GRK4. Identification of four splice variants.

A novel human G protein-coupled receptor kinase was recently identified by positional cloning in the search for the Huntington's disease locus (Ambrose, C., James, M., Barnes, G., Lin, C., Bates, G., Altherr, M., Duyao, M., Groot, N., Church, D., Wasmuth, J. J., Lehrach, H., Housman, D., Buckler, A., Gusella, J. F., and MacDonald, M. E. (1993) Hum. Mol. Genet. 1, 697-703). Comparison of the deduced amino acid sequence of GRK4 with those of the closely related GRK5 and GRK6 suggested the apparent loss of 32 codons in the amino-terminal domain and 46 codons in the carboxyl-terminal domain of GRK4. These two regions undergo alternative splicing in the GRK4 mRNA, resulting from the presence or absence of exons filling one or both of these apparent gaps. Each inserted sequence maintains the open reading frame, and the deduced amino acid sequences are similar to corresponding regions of GRK5 and GRK6. Thus, the GRK4 mRNA and the GRK4 protein can exist as four distinct variant forms. The human GRK4 gene is composed of 16 exons extending over 75 kilobase pairs of DNA. The two alternatively spliced exons correspond to exons II and XV. The genomic organization of the GRK4 gene is completely distinct from that of the human GRK2 gene, highlighting the evolutionary distance since the divergence of these two genes. Human GRK4 mRNA is expressed highly only in testis, and both alternative exons are abundant in testis mRNA. The four GRK4 proteins have been expressed, and all incorporate [3H]palmitate. GRK4 is capable of augmenting the desensitization of the rat luteinizing hormone/chorionic gonadotropin receptor upon coexpression in HEK293 cells and of phosphorylating the agonist-occupied, purified beta2-adrenergic receptor, indicating that GRK4 is a functional protein kinase.

Analysis of the dopamine receptor family in the compact genome of the puffer fish Fugu rubripes.

The genome of the puffer fish, Fugu rubripes (Fugu), is approximately 400 Mb, 7.5 times smaller than that of human. We have isolated four dopamine receptor-like genes from Fugu genomic DNA. These genes show high sequence and structural homology to the known dopamine receptor genes, although, in contrast to previously described genes from this species, the intron size is comparable to that in human. The 5' noncoding exons of the D2/D3 dopamine receptor-like genes is described and compared to that in human. The high gene density of Fugu is shown by the close proximity of a cystatin-like gene 1503 bp from the dopamine receptor gene D222. We propose that the compact genome of Fugu can serve as a model for identifying gene family members directly from genomic DNA.

One armed PCR (OA-PCR): amplification of genomic DNA from a single primer domain.

One-armed PCR (OA-PCR) is a novel technique that allows amplification of genomic DNA from a single region of known sequence. Previously described methods are either limited to cDNA or require extensive manipulation of template prior to amplification. In OA-PCR, DNA from a small insert phage Fugu genomic library is amplified using two sequence-specific primers and a tailed vector primer. Single, specific products of between 70 bp and 1.9 kb (mean 600 bp) have been obtained following two rounds of amplification and directly sequenced without cloning. The optimum parameters have been investigated: primer pairs of 18-24 bp, separated by between 0 and 500 bp, have successfully amplified a specific product. OA-PCR represents a rapid, simple method to extend genomic sequence into noncoding and regulatory sequences. In addition, degenerate OA-PCR primer pairs have allowed cross-species amplification of novel Fugu gene homologues.

Endothelin an endocrine role.

Endothelin is a potent and long-acting vasoconstrictor first isolated from the vascular endothelium. It was found in the search for the long-postulated endothelial vascular smooth muscle activator. Recent reports, however, suggest that endothelin may affect the release of the pituitary hormones and control the levels of atrial natriuretic peptide, renin, and the catecholamines. Its widespread distribution within the CNS and other tissues suggests that endothelin may have an important function as a neurotransmitter.

Very high concentrations of islet amyloid polypeptide are necessary to alter the insulin response to intravenous glucose in man.

Islet amyloid polypeptide (IAPP) is a beta-cell peptide that can oppose insulin action in animal systems, but has not been shown to have any action in man; previously, we failed to show an effect of infused IAPP on iv glucose tolerance in human volunteers. We have reexamined its effects at even higher concentrations in six volunteers who received iv glucose (0.5 g/kg) during infusions of IAPP (25 and 50 pmol/kg.min) or normal saline. IAPP rose from a mean basal of 14.7 +/- 5.3 pmol/L to peak levels of 1,420 +/- 110, 2,240 +/- 140, and 27.7 +/- 9 pmol/L, respectively. IAPP at 25 pmol/kg.min had no effect on the plasma glucose disposal rate or the total incremental insulin response, but, in contrast, at 50 pmol/kg.min decreased the insulin response to glucose compared to the saline infusion (incremental area under the curve, 11,276 +/- 2,353 vs. 17,549 +/- 2,687 U; mean +/- SEM; P less than 0.02). This decrease was observed both during the first phase (0-10 min postglucose) insulin response (3,210 +/- 985 vs. 4,382 +/- 815 U; P less than 0.05) and the second phase response (11-90 min, 8,520 +/- 1,719 vs. 13,679 +/- 2,326 U; P less than 0.03). Glucose disposal rate, however, was unaffected (2.0 +/- 0.2 vs. 1.9 +/- 0.2). Thus, circulating IAPP concentrations greater than 90 times normal postprandial peaks were necessary to affect the insulin response to glucose. IAPP appears unlikely to be a circulating hormone influencing carbohydrate metabolism in man.

Diaphragmatic weakness in hereditary motor and sensory neuropathy.

Six patients severely affected by hereditary motor and sensory neuropathy (HMSN), four type I and two type II, had clinical evidence of diaphragmatic weakness. One presented with cardiorespiratory failure secondary to nocturnal hypoventilation, and three others were unable to lie flat because of dyspnoea. Diaphragmatic paralysis should be considered as a cause of respiratory or cardiac failure in cases of HMSN.

Assessment of postural hypotension in elderly patients.

Change in blood pressure on standing was measured in 23 geriatric inpatients during the morning. The fall in systolic blood pressure was found to be greatest 30 s after standing, at 9.3 +/- 3.3 (SEM) mmHg and had returned to the supine levels within 2 min. In contrast, the diastolic pressure rose to a maximum of 9.7 +/- 1.8 mmHg by 2 min. In 13 patients the measurements were repeated in the afternoon after lunch. The systolic blood pressure drop was significantly greater: 20.8 +/- 3.6 against 7.1 +/- 2.0 mmHg in the morning (p = 0.01). Standardization of the time after standing and time of day of measurement may allow more precise comparisons of different studies.

The rise and fall of Legionnaires' disease in Nottingham.

The annual number of confirmed cases of Legionnaires' disease in both Nottingham, and England and Wales, reached a peak in 1980 and has since declined. Legionella infection is a rare cause of community-acquired pneumonia managed at home (accounting for less than 1% of cases), more common in those admitted to hospital (5-15%) and more common still in patients with severe pneumonia as seen on an intensive care unit (up to 30% of cases). Antibiotic therapy for any patient with moderate or severe pneumonia of uncertain aetiology should cover legionella infection.

Differential blocking actions of idazoxan against the inhibitory effects of 6-fluoronoradrenaline and clonidine in the rat vas deferens.

The prejunctional inhibitory effects of clonidine and 6-fluoronoradrenaline (6-FNA) have been evaluated in the isolated prostatic segment of the rat vas deferens, against the twitch response evoked by low frequency (0.1 Hz) field stimulation. The inhibitory potency of 6FNA was significantly increased in the presence of cocaine (1 microM) or pargyline (10 microM), but was not modified in the vas deferens from rats pretreated with reserpine when the endogenous levels of noradrenaline (NA) were decreased by 97%. Clonidine was significantly more potent than 6-FNA as an inhibitory agonist, and the potency of clonidine was not modified after cocaine, pargyline or reserpine. The alpha 2-adrenoceptor blocking agent idazoxan, was a competitive antagonist against the inhibitory effects of clonidine under all experimental conditions. In contrast, the only antagonism shown by idazoxan against the inhibitory effects of 6-FNA was in the presence of cocaine (1 microM), and this antagonist effect of idazoxan was not concentration-related. Low concentrations of 6-FNA caused concentration-dependent facilitatory effects on the twitch response, which were significantly greater after treatment with idazoxan (1 microM) in reserpine-treated vas deferens. These facilitatory effects of 6-FNA were always observed in the presence of prazosin (300 nM) and also after treatment of the preparations with phenoxybenzamine (10 microM), a concentration which abolished the inhibitory actions of both clonidine and 6-FNA. The facilitatory effects on the twitch response induced by low concentrations of 6-FNA are therefore unlikely to be due to either alpha 1- or alpha 2-adrenoceptor stimulation. In conclusion, the failure of idazoxan to block the inhibitory effects of 6-FNA, while exerting a potent competitive antagonism of clonidine-induced inhibitory effects, supports the proposal that alpha 2-adrenoceptors may in fact be subdivided into two subclasses, involving imidazoline and phenylethylamine recognition sites.

Aetiology and outcome of severe community-acquired pneumonia.

Between January 1972 and December 1981, 50 patients with severe community-acquired pneumonia were admitted to the intensive care unit of a district general hospital. A causal pathogen was identified in 41 cases (82%). Streptococcus pneumoniae (16 cases), Legionella pneumophila (15 cases) and Staphylococcus aureus (5 cases) were the commonest. Assisted ventilation was required in 44 patients, of whom 25 died (57%). All 5 patients with staphylococcal pneumonia and 12(75%) with pneumococcal pneumonia died. Only 5 (33%) with Legionnaires' disease died. Mortality was significantly associated with age. Recommendations for the management of severe pneumonia are made.

Laboratory diagnosis of smallpox: role of the Virus Reference Laboratory, Colindale, 1947-70.

The Virus Reference Laboratory, Colindale, first embarked on laboratory investigations for smallpox early in 1947. From then, in conjunction with the Department of Bacteriology, University of Liverpool, it provided a complete diagnostic service throughout England and Wales until 1962, after which the service became available regionally until eradication was effected. Up to 1970 it had investigated 2696 specimens from suspected cases of smallpox and had recovered 108 strains of variola and 248 of vaccinia virus. These last were from persons suffering the complications of vaccination. Some outbreaks following smallpox importation are discussed but infection among laboratory staff during this period was not demonstrated.

Hospital study of adult community-acquired pneumonia.

The cause of primary pneumonia was diagnosed in 124 of 127 consecutive adult patients admitted to hospital with community-acquired illness. Pneumococcal infection was found in 96 (76%) patients and legionnaries' disease was the second commonest infection identified (15%). Other bacterial infections were uncommon. 11 patients had atypical pneumonia, including 7 with psittacosis. There were several mixed infections and most of the 11 patients with viral infections also had bacterial pneumonia. 19 patients died (15%) and mortality was associated with increasing age, the presence of coexisting disease, and the cause of the pneumonia. Recognition of the most likely causes of severe pneumonia allows logical initial antibiotic treatment for such patients admitted to hospital.