Opiates such as heroin and morphine and the endogenous opioid neuropeptides are potent modulators of immune function. There is evidence linking opiate abuse with immune suppression and this factor may be important in both the symptomology and progression of HIV disease. Many studies have catalogued opiate/opioid effects on immune cells; however, the underlying mechanisms by which these agents modulate immune functions are largely unknown. We have initiated studies to identify the lymphocyte receptors for opiates/opioids and to define the mechanisms by which they regulate immune cell functions. We have identified lymphocyte opiate receptors and measured their numbers and affinity. We have shown these receptors to be functional and to deliver transmembrane signals that elicit increases in both intracellular, free Ca2+ and cAMP. To gain an understanding of the molecular properties of these proteins, and ultimately, of how opioids regulate lymphocyte functions we have now initiated studies with a novel human lymphocyte cDNA clone that codes for an opioid-binding protein. This cDNA was isolated from a human lymphocyte library using subtractive hybridization methods. Upon inspection, it was found to belong to the 6 protein-coupled superfamily of membrane bound receptors that have seven membrane-spanning domains (R7G). We have shown (l) specific binding of 3H-DADLE to membranes from COS cells transfected with the cDNA encoding this novel protein and (2) functionality, in that activation of transfected COS cells with methionine-enkephalin (MET-ENK) results in an increase in the intracellular concentration of cAMP. We propose to define the pharmacological specificity of the binding site and correlate receptor-ligand binding with activity using COS cells transfected with cDNA encoding the lymphocyte protein. We propose to identify G-protein regulatory subunits that associate with the lymphocyte protein by using opiate/opioid agonist-stimulated incorporation of the photoreactive GTP analog, azidoanilido[alpha-32P]GTP. Binding and signaling domains of the protein will be mapped with both mutant and wild- type DNAs expressed in COS cells. The key structural residues and domains will be identified; ligand-receptor binding and activity will be correlated with sequence analyses. Mutant proteins will be designed using site-directed mutagenesis in vitro. We will define the distribution of the novel lymphocyte R7G in a well characterized panel of lymphoid cells having defined phenotypes and biological properties, as well as in normal T and B cells. We will measure receptor expression at both the mRNA and surface protein levels. Finally, the effects of chronic exposure to opiate/opioids on the expression and signaling of the lymphocyte R7G will be defined.
| Heagy, W; Teng, E; Lopez, P et al. (1999) Enkephalin receptors and receptor-mediated signal transduction in cultured human lymphocytes. Cell Immunol 191:34-48 |
